Biology subjects

Filippov, D. V.

Publications and source records attributed to Filippov, D. V..

6 recordsLinked to original sources

bioRxiv2026

Identification of novel cellular intermediates unveils unique enzymes for flagellar glycan biosynthesis in Clostridioides difficile

Glycosylation of bacterial surface proteins, such as flagellin (FliC), is important for their function and is often involved in virulence of pathogens. Glycans can be further modified by so-called post-glycosylation modifications (PGMs) often resulting in exclusive molecular structures. In Clostridioides difficile a unique glycan structure (Type A) decorates FliC (which forms the flagellar filament) that consists of an O-linked N-acetyl-{beta}-D-glucosamine (GlcNAc) modified with an N-methyl-L-threonine via a phosphodiester linkage. This PGM is synthesized by a set of four enzymes encoded in one operon (ftaABCD), but the exact biosynthesis pathway and biosynthetic intermediates remain unknown. In this study, we chemically synthesized two hitherto undescribed biosynthetic intermediates that we predicted based on bioinformatic analyses, CDP-threonine and CDP-N-methylthreonine. We showed that they are involved in the Type A PGM biosynthesis, as evidenced by mass spectrometric analyses of extracts of a set of C. difficile mutant strains. Furthermore, we characterized FtaC to be a SAM-dependent CDP-threonine N-methyltransferase, that installs the methyl group on CDP-threonine prior to transfer of the PGM to GlcNAc-FliC, and we revealed FtaD as the CDP-N-methylthreonine:GlcNAc N-methylthreoninephosphotransferase. Finally, using recombinantly expressed FtaC and FtaD in combination with synthetic CDP-threonine, we reconstituted the biosynthesis pathway of the Type A PGM in vitro. Overall, our results open avenues to explore these unique biosynthesis enzymes in molecular detail to provide new points of entry for the development of biosynthesis inhibitors and tools to study the role of this PGM in virulence and flagellar assembly.

microbiology↗

bioRxiv2026

PARP1 recruits SPRTN to DNA-protein crosslinks through a conserved poly-ADP-ribose binding domain.

DNA-protein crosslinks (DPCs) are toxic DNA lesions formed by the covalent attachment of proteins to DNA. Failure to resolve DPCs leads to genomic instability, premature aging, and cancer predisposition. Although multiple proteases and the 26S proteasome degrade DPCs, how these lesions are detected and marked for proteolysis remains unclear. Here, we show that poly-(ADP-ribose) polymerases (PARP1/2) sense DPCs and modify them with poly(ADP-ribose) (PAR) to promote repair via a SPRTN-Tdp1 axis. We discovered a Nudix homology domain (NHD) in SPRTN that mediates direct non-covalent PAR binding and is important for DPC repair. Loss of PARP1/2 activity or mutation of the SPRTN NHD leads to sustained DPCs. Single-molecule analysis revealed that SPRTN does not bind efficiently to the DPC, however after the addition of PARP1 in the presence of NAD+, SPRTN binding to the DPC was significantly increased. Our findings establish PARP1/2 enzymes as immediate DPC sensors, reveal PARylation as a signal marking DPCs for SPRTN-dependent degradation, and identify SPRTN as the first PARP-directed protease.

molecular biology↗

bioRxiv2024

Parp7 generates an ADP-ribosyl degron that controls negative feedback of androgen signaling

AbstractThe androgen receptor (AR) tranduces the effects of circulating and tumor-derived androgens to the nucleus through ligand-induced changes in protein conformation, localization, and engagement with chromatin binding sites. Understanding these events and their integration with signal transduction is critical for defining how AR drives prostate cancer and unveiling pathway features that are amenable to therapeutic intervention. Here, we describe a novel post-transcriptional mechanism that controls AR protein levels on chromatin and associated gene output which is based on a highly selective, inducible degradation mechanism. We find that the mono-ADP-ribosyltransferase PARP7 generates an ADP-ribosyl degron on a single cysteine within the DNA binding domain of AR, which is then recognized by the ADP- ribose reader domain in the ubiquitin E3 ligase DTX2 and degraded by the proteasome. Mathematical modeling of the pathway suggested that PARP7 ADP-ribosylates chromatin-bound AR, a prediction that was validated in cells using an AR mutant that undergoes nuclear import but fails to bind DNA. Lysine- independent, non-conventional ubiquitin conjugation to ADP-ribosyl-cysteine and AR degradation by the proteasome forms the basis of a negative feedback loop that regulates specific modules of AR target genes. Our data expand the repertoire of mono-ADP-ribosyltransferase enzymes to include gene regulation based on highly selective protein degradation. One Sentence SummaryPARP7 mono-ADP-ribosylates the androgen receptor on Cys620 to mark the androgen receptor for ubiquitin conjugation by an E3 ligase with ADP-ribose reader function, resulting in in negative feedback of AR-dependent gene expression.

cancer biology↗

bioRxiv2023

PARP14 is a PARP with both ADP-ribosyl transferase and hydrolase activities

PARP14 is a mono-ADP-ribosyl transferase involved in the control of immunity, transcription and DNA replication stress management. However, little is known about the ADP-ribosylation activity of PARP14, including its substrate specificity or how PARP14-dependent ADP-ribosylation is reversed. Here we show that PARP14 is dual function enzyme with both ADP-ribosyl transferase and hydrolase activity acting on both protein and nucleic acid substrates. In particular, we show that the PARP14 macrodomain 1 is an active ADP-ribosyl hydrolase. We also demonstrate hydrolytic activity for the first macrodomain of PARP9. We reveal that expression of a PARP14 mutant with the inactivated macrodomain 1 results in a dramatic increase in mono(ADP-ribosyl)ation of proteins in human cells, including PARP14 itself and antiviral PARP13. Moreover, we demonstrate that the closely related hydrolytically active macrodomain of SARS2 Nsp3, Mac1, efficiently reverses PARP14 ADP-ribosylation in vitro and in cells, supporting the evolution of viral macrodomains to counteract PARP14-mediated antiviral response. TeaserPARP14 is an antiviral PARP that combines ADP-ribosylation writer, reader and eraser functions in one polypeptide.

cell biology↗

bioRxiv2022

Systematic analysis of ADP-ribose detection reagents and optimisation of sample preparation to detect ADP-ribosylation in vitro and in cells

Recent evidence suggests that modification of substrates with a single ADP-ribose (ADPr) is important in for example antiviral immunity and cancer. However, the endogenous substrates and the extent of mono-ADP-ribosylation are still largely unclear. Several reagents were developed to detect ADP-ribosylation but it is unknown whether they recognise only ADPr, amino acid-ADPr linkages or a combination of ADPr with a protein backbone. We screened the affinity of selected reagents for enzymatically, chemically and in cell generated ADP-ribosylation on glutamate, cysteine, serine, arginine, threonine and RNA by blotting, as well as analysed the subcellular sites of ADP-ribosylation using immunofluorescence confocal microscopy. We furthermore observed that the modification is heat-labile and optimised sample preparation procedures. Our comparison of the available reagents, as well as optimisation of sample preparation, will allow future work further dissecting the function of ADP-ribosylation in cells, both on protein and on RNA substrates.

molecular biology↗

bioRxiv2017

Qinichelins, novel catecholate-hydroxamate siderophores synthesized via a multiplexed convergent biosynthesis pathway

The explosive increase in genome sequencing and the advances in bioinformatic tools have revolutionized the rationale for natural product discovery from actinomycetes. In particular, this has revealed that actinomycete genomes contain numerous orphan gene clusters that have the potential to specify many yet unknown bioactive specialized metabolites, representing a huge unexploited pool of chemical diversity. Here, we describe the discovery of a novel group of catecholate-hydroxamate siderophores termed qinichelins (2-5) from Streptomyces sp. MBT76. Correlation between the metabolite levels and the protein expression profiles identified the biosynthetic gene cluster (BGC; named qch) most likely responsible for qinichelin biosynthesis. The structure of the molecules was elucidated by bioinformatics, mass spectrometry and NMR. Synthesis of the qinichelins requires the interplay between four gene clusters, for its synthesis and for precursor supply. This biosynthetic complexity provides new insights into the challenges scientists face when applying synthetic biology approaches for natural product discovery.\n\nPride repository reviewer account details:\n\nURL: https://www.ebi.ac.uk/pride/archive/login\n\nProject accession: PXD006577\n\nUsername: reviewer35793@ebi.ac.uk\n\nPassword: 3H0iM1FK

microbiology↗