Stepsiblings of the PARP family: modulation of ADPribosylation by HPF1 and DTX E3 ligases

International audienceI will start my talk by introducing protein posttranslational modifications (PTMs) and the PARP protein family. Then, I will discuss selected proteins related to PARPs: the HPF1 cofactor and the DTX family of E3 ubiquitin ligases. I will build on my work on these two systems performed together with colleagues from the Ivan Ahel group in Oxford. HPF1 forms a complex with either PARP1 or PARP2, creating a composite enzyme that efficiently catalyses protein serine ADPribosylation. Within this complex, HPF1 recognises and activates the serine residue of a protein substrate. The DTX E3 ligases work in conjunction with E2 enzymes to catalyse a unique ubiquitylation reaction on the hydroxyl group of an ADPribose moiety. Like HPF1, DTX ligases seem to activate an acceptor of a modification reaction. The reaction catalyzed by DTX:E2 complexes could generate hybrid ADPriboseubiquitin signals attached to proteins or other substrates. My talk will focus on mechanistic, structural, and evolutionary principles while also discussing the biological implications of these findings

Bridging of DNA breaks activates PARP2–HPF1 to modify chromatin

Breaks in DNA strands recruit the protein PARP1 and its paralogue PARP2 to modify histones and other substrates through the addition of mono- and poly(ADP-ribose) (PAR)1,2,3,4,5. In the DNA damage responses, this post-translational modification occurs predominantly on serine residues6,7,8 and requires HPF1, an accessory factor that switches the amino acid specificity of PARP1 and PARP2 from aspartate or glutamate to serine9,10. Poly(ADP) ribosylation (PARylation) is important for subsequent chromatin decompaction and provides an anchor for the recruitment of downstream signalling and repair factors to the sites of DNA breaks2,11. Here, to understand the molecular mechanism by which PARP enzymes recognize DNA breaks within chromatin, we determined the cryo-electron-microscopic structure of human PARP2–HPF1 bound to a nucleosome. This showed that PARP2–HPF1 bridges two nucleosomes, with the broken DNA aligned in a position suitable for ligation, revealing the initial step in the repair of double-strand DNA breaks. The bridging induces structural changes in PARP2 that signal the recognition of a DNA break to the catalytic domain, which licenses HPF1 binding and PARP2 activation. Our data suggest that active PARP2 cycles through different conformational states to exchange NAD+ and substrate, which may enable PARP enzymes to act processively while bound to chromatin. The processes of PARP activation and the PARP catalytic cycle we describe can explain mechanisms of resistance to PARP inhibitors and will aid the development of better inhibitors as cancer treatments12,13,14,15,16

ADP-ribosylation from molecular mechanisms to therapeutic implications

International audienceADP-ribosylation is a ubiquitous modification of biomolecules, including proteins and nucleic acids, that regulates various cellular functions in all kingdoms of life. The recent emergence of new technologies to study ADP-ribosylation has reshaped our understanding of the molecular mechanisms that govern the establishment, removal, and recognition of this modification, as well as its impact on cellular and organismal function. These advances have also revealed the intricate involvement of ADP-ribosylation in human physiology and pathology and the enormous potential that their manipulation holds for therapy. In this review, we present the state-of-the-art findings covering the work in structural biology, biochemistry, cell biology, and clinical aspects of ADP-ribosylation

New insights into the catalysis of modification reactions

International audienceIn this presentation, I will share our recent work on two modification reactions that are catalysed through a new paradigm. This paradigm involves a composite active site composed of catalytic residues from two different proteins. First, I will talk about how PARP1, a well-characterised ADP-ribosylating enzyme, is complemented by the protein cofactor HPF1. HPF1 is required for recognising and activating a serine residue in a protein substrate. In the second part, I will discuss an analogous interaction between DELTEX E3 ligases and E2 enzymes in catalysing a novel ubiquitylation reaction on a hydroxyl group of an ADP-ribose moiety. The talk will highlight chemical and mechanistic analogies between different modification reactions.References:[1] MJ Suskiewicz, F Zobel, T Ogden, …, D. Neuhaus, I. Ahel, Nature, 2020, 579, 598-602[2] K Zhu, MJ Suskiewicz, …, V. Aucagne, D. Ahel, I. Ahel, Sci Adv, 2022, 8(40):eadd425

The potential of PARP inhibitors in targeted cancer therapy and immunotherapy

International audienceDNA damage response (DDR) deficiencies result in genome instability, which is one of the hallmarks of cancer. Poly (ADP-ribose) polymerase (PARP) enzymes take part in various DDR pathways, determining cell fate in the wake of DNA damage. PARPs are readily druggable and PARP inhibitors (PARPi) against the main DDR-associated PARPs, PARP1 and PARP2, are currently approved for the treatment of a range of tumor types. Inhibition of efficient PARP1/2-dependent DDR is fatal for tumor cells with homologous recombination deficiencies (HRD), especially defects in breast cancer type 1 susceptibility protein 1 or 2 (BRCA1/2)dependent pathway, while allowing healthy cells to survive. Moreover, PARPi indirectly influence the tumor microenvironment by increasing genomic instability, immune pathway activation and PD-L1 expression on cancer cells. For this reason, PARPi might enhance sensitivity to immune checkpoint inhibitors (ICIs), such as anti-PD-(L)1 or anti-CTLA4, providing a rationale for PARPi-ICI combination therapies. In this review, we discuss the complex background of the different roles of PARP1/2 in the cell and summarize the basics of how PARPi work from bench to bedside. Furthermore, we detail the early data of ongoing clinical trials indicating the synergistic effect of PARPi and ICIs. We also introduce the diagnostic tools for therapy development and discuss the future perspectives and limitations of this approach

DELTEX E3 ligases ubiquitylate ADP-ribosyl modification on nucleic acids

International audienceAlthough ubiquitylation had traditionally been considered limited to proteins, the disco v ery of non-proteinaceous substrates (e.g. lipopolysaccharides and adenosine diphosphate ribose (ADPr)) challenged this perspective. Our recent study sho w ed that DTX2 E3 ligase efficiently ubiquitylates ADP r. Here, w e sho w that the ADP r ubiquit ylation activit y is also present in another DELTEX family member, DTX3L, analysed both as an isolated catalytic fragment and the full-length PARP9:DTX3L complex, suggesting that it is a general feature of the DELTEX family. Since str uct ural predictions show that DTX3L possesses single-stranded nucleic acids binding ability and given the fact that nucleic acids have recently emerged as substrates for ADP-ribosylation, we asked whether DELTEX E3s might cat alyse ubiquit ylation of an ADPr moiety linked to nucleic acids. Indeed, w e sho w that DTX3L and DTX2 are capable of ubiquitylating ADP-ribosylated DNA and RNA synthesized by PARPs, including PARP14. Furthermore, we demonstrate that the Ub-ADPr-nucleic acids conjugate can be reversed by two groups of hydrolases, which remove either the whole adduct (e.g. SARS-CoV-2 Mac1 or PARP14 macrodomain 1) or just the Ub (e.g. SARS-CoV-2 PLpro). Overall, this study reveals ADPr ubiquitylation as a general function of the DELTEX family E3s and presents the evidence of re v ersible ubiquitylation of ADP-ribosylated nucleic acids

Structure of McsB, a protein kinase for regulated arginine phosphorylation

Protein phosphorylation regulates key processes in all organisms. In Gram-positive bacteria, protein arginine phosphorylation plays a central role in protein quality control by regulating transcription factors and marking aberrant proteins for degradation. Here, we report structural, biochemical, and in vivo data of the responsible kinase, McsB, the founding member of an arginine-specific class of protein kinases. McsB differs in structure and mechanism from protein kinases that act on serine, threonine, and tyrosine residues and instead has a catalytic domain related to that of phosphagen kinases (PhKs), metabolic enzymes that phosphorylate small guanidino compounds. In McsB, the PhK-like phosphotransferase domain is structurally adapted to target protein substrates and is accompanied by a novel phosphoarginine (pArg)-binding domain that allosterically controls protein kinase activity. The identification of distinct pArg reader domains in this study points to a remarkably complex signaling system, thus challenging simplistic views of bacterial protein phosphorylation

Serine ADP-ribosylation in Drosophila provides insights into the evolution of reversible ADP-ribosylation signalling

International audienceIn the mammalian DNA damage response, ADP-ribosylation signalling is of crucial importance to mark sites of DNA damage as well as recruit and regulate repairs factors. Specifically, the PARP1:HPF1 complex recognises damaged DNA and catalyses the formation of serine-linked ADP-ribosylation marks (mono-Ser-ADPr), which are extended into ADP-ribose polymers (poly-Ser-ADPr) by PARP1 alone. Poly-Ser-ADPr is reversed by PARG, while the terminal mono-Ser-ADPr is removed by ARH3. Despite its significance and apparent evolutionary conservation, little is known about ADP-ribosylation signalling in non-mammalian Animalia . The presence of HPF1, but absence of ARH3, in some insect genomes, including Drosophila species, raises questions regarding the existence and reversal of serine-ADP-ribosylation in these species. Here we show by quantitative proteomics that Ser-ADPr is the major form of ADP-ribosylation in the DNA damage response of Drosophila melanogaster and is dependent on the d Parp1: d Hpf1 complex. Moreover, our structural and biochemical investigations uncover the mechanism of mono-Ser-ADPr removal by Drosophila Parg. Collectively, our data reveal PARP:HPF1-mediated Ser-ADPr as a defining feature of the DDR in Animalia . The striking conservation within this kingdom suggests that organisms that carry only a core set of ADP-ribosyl metabolising enzymes, such as Drosophila , are valuable model organisms to study the physiological role of Ser-ADPr signalling

Serine ADP-ribosylation in Drosophila provides insights into the evolution of reversible ADP-ribosylation signalling

Abstract In the mammalian DNA damage response, ADP-ribosylation signalling is of crucial importance to mark sites of DNA damage as well as recruit and regulate repairs factors. Specifically, the PARP1:HPF1 complex recognises damaged DNA and catalyses the formation of serine-linked ADP-ribosylation marks (mono-Ser-ADPr), which are extended into ADP-ribose polymers (poly-Ser-ADPr) by PARP1 alone. Poly-Ser-ADPr is reversed by PARG, while the terminal mono-Ser-ADPr is removed by ARH3. Despite its significance and apparent evolutionary conservation, little is known about ADP-ribosylation signalling in non-mammalian Animalia. The presence of HPF1, but absence of ARH3, in some insect genomes, including Drosophila species, raises questions regarding the existence and reversal of serine-ADP-ribosylation in these species. Here we show by quantitative proteomics that Ser-ADPr is the major form of ADP-ribosylation in the DNA damage response of Drosophila melanogaster and is dependent on the dParp1:dHpf1 complex. Moreover, our structural and biochemical investigations uncover the mechanism of mono-Ser-ADPr removal by Drosophila Parg. Collectively, our data reveal PARP:HPF1-mediated Ser-ADPr as a defining feature of the DDR in Animalia. The striking conservation within this kingdom suggests that organisms that carry only a core set of ADP-ribosyl metabolising enzymes, such as Drosophila, are valuable model organisms to study the physiological role of Ser-ADPr signalling