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ADP-ribosylation is a pivotal post-translational modification that mediates various important cellular processes producing negatively charged biopolymer, poly (ADP-ribose), the functions of which need further elucidation. Toward this end, the availability of well-defined ADP-ribose (ADPr) oligomers in sufficient quantities is a necessity. In this work, we demonstrate the chemical synthesis of linear ADPr oligomers of defined, increasing length using a modified solid phase synthesis method. An advanced phosphoramidite building block temporarily protected with the base sensitive Fm-group was designed and implemented in the repeating pyrophosphate formation via a P(v)–P(iii) coupling procedure on Tentagel solid support. Linear ADPr oligomers up to a pentamer were successfully synthesized and their affinity for the poly-(ADP-ribose)-binding macrodomain of the human oncogenic helicase and chromatin remodeling enzyme ALC1 was determined. Our data reveal a length-dependent binding manner of the nucleic acid, with larger ADPr oligomers exhibiting higher binding enthalpies for ALC1, illustrating how the activity of this molecular machine is gated by PAR., We report the synthesis of linear ADPr oligomers of defined length up to a pentamer using an improved solid phase method. Binding study with human oncogenic helicase ALC1 shows that ADPr oligomers bind to ALC1 in a length-dependent manner.

Pyridoxal 5′‐phosphate (PLP) is an essential cofactor for neurotransmitter metabolism. Pyridoxal phosphatase (PDXP) deficiency in mice increases PLP and γ‐aminobutyric acid levels in the brain, yet how PDXP is regulated is unclear. Here, we identify the Ca\(^{2+}\)‐ and integrin‐binding protein 1 (CIB1) as a PDXP interactor by yeast two‐hybrid screening and find a calmodulin (CaM)‐binding motif that overlaps with the PDXP‐CIB1 interaction site. Pulldown and crosslinking assays with purified proteins demonstrate that PDXP directly binds to CIB1 or CaM. CIB1 or CaM does not alter PDXP phosphatase activity. However, elevated Ca\(^{2+}\) concentrations promote CaM binding and, thereby, diminish CIB1 binding to PDXP, as both interactors bind in a mutually exclusive way. Hence, the PDXP‐CIB1 complex may functionally differ from the PDXP‐Ca\(^{2+}\)‐CaM complex.

NAD metabolism is essential for all forms of life. Compartmental regulation of NAD consumption, especially between the nucleus and the mitochondria, is required for energy homeostasis. However, how compartmental regulation evolved remains unclear. In the present study, we investigated the evolution of the macrodomain-containing histone variant macroH2A1.1, an integral chromatin component that limits nuclear NAD consumption by inhibiting poly(ADP-ribose) polymerase 1 in vertebrate cells. We found that macroH2A originated in premetazoan protists. The crystal structure of the macroH2A macrodomain from the protist Capsaspora owczarzaki allowed us to identify highly conserved principles of ligand binding and pinpoint key residue substitutions, selected for during the evolution of the vertebrate stem lineage. Metabolic characterization of the Capsaspora lifecycle suggested that the metabolic function of macroH2A was associated with nonproliferative stages. Taken together, we provide insight into the evolution of a chromatin element involved in compartmental NAD regulation, relevant for understanding its metabolism and potential therapeutic applications., For technical support, we thank the research service facilities of IJC and IGTP, the Crystallization Facility of the Max Planck Institute of Biochemistry, the ICTS NMR facility from the Scientific and Technological Centres of the University of Barcelona and Biophysics Core Facility of BMC-LMU. I.G. was a fellow of the Marie Skłodowska Curie Training network ‘ChroMe’ (H2020-MSCA-ITN-2015-675610, awarded to M.B. and A.G.L.). The project was further supported by national grants (nos. RTI2018-094005-B-I00 and BFU2015-66559-P from FEDER/Ministerio de Ciencia e Innovación—Agencia Estatal de Investigación to M.B.). Research in the participating labs was further supported by the following grants: the Marie Skłodowska Curie Training network ‘INTERCEPT-MDS’ no. H2020-MSCA-ITN-2020-953407 (to M.B.), MINECO-ISCIII no. PIE16/00011 (to M.B.); the Deutsche José Carreras Leukämie Stiftung DJCLS (no. 14R/2018 to M.B.), AGAUR (no. 2017-SGR-305 to M.B.), Fundació La Marató de TV3 (no. 257/C/2019 to M.B.), German Research Foundation Project (ID 213249687—SFB 1064 and Project ID 325871075—SFB 1309 to A.G.L.), the Spanish Ministry of Science (PID2019-110183RB-C21 to A.R.M.), Community of Madrid (P2018/BAA-4343-ALIBIRD2020-CM to A.R.M), Ramón Areces Foundation (to A.R.M.), National Science Foundation (EF-1921402 to J.M.E.L.), 2015 International Doctoral Fellowship La Caixa-Severo Ochoa (to M.F.V.), Marie Skłodowska-Curie Individual Fellowship (no. 747789 to M.M.L.), Juan de la Cierva-Incorporación (IJC2018-036657-I to M.M.L., ERC-2012-CoG-616960 to I.R.T.), MINECO (BFU2017-90114-P to I.R.T.), AGAUR (2017-SGR-324 to X.S.) and MINECO (BIO2015-70092-R and ERC-2014-CoG-648201 to X.S.). Research at the IJC is supported by the ‘La Caixa’ Foundation, Fundació Internacional Josep Carreras, Celgene Spain and the CERCA Programme/Generalitat de Catalunya.

Chromatin remodeling enzymes are large molecular machines that guard the genome by reorganizing chromatin structure. They can reposition, space and evict nucleosomes and thus control gene expression, DNA replication and repair. Recent cryo-electron microscopy (cryo-EM) analyses have captured snapshots of various chromatin remodelers as they interact with nucleosomes. In this review, we summarize and discuss the advances made in our understanding of the regulation of chromatin remodelers, the mode of DNA translocation, as well as the influence of associated protein domains and remodeler subunits on the specific functions of chromatin remodeling complexes. The emerging structural information will help our understanding of disease mechanisms and guide our knowledge toward innovative therapeutic interventions. Copyright © 2020 Elsevier Ltd. All rights reserved.

Background Poly-ADP-ribose polymerases (PARPs) are key mediators of cellular stress response. They are intimately linked to cellular metabolism through the consumption of NAD+. PARP1/ARTD1 in the nucleus is the major NAD+ consuming activity and plays a key role in maintaining genomic integrity. Scope of review In this review, we discuss how different organelles are linked through NAD+ metabolism and how PARP1 activation in the nucleus can impact the function of distant organelles. We discuss how differentiated cells tame PARP1 function by upregulating an endogenous inhibitor, the histone variant macroH2A1.1. Major conclusions The presence of macroH2A1.1, particularly in differentiated cells, raises the threshold for the activation of PARP1 with consequences for DNA repair, gene transcription, and NAD+ homeostasis., Highlights • Beyond DNA repair, PARP1 is essential for metabolic homeostasis. • Epigenetic mechanisms prevent metabolic disorders through PARP1 taming. • Beyond cancer, the development of PARP1 inhibitors offers diverse clinical potential.

Background: Poly-ADP-ribose polymerases (PARPs) are key mediators of cellular stress response. They are intimately linked to cellular metabolism through the consumption of NAD+. PARP1/ARTD1 in the nucleus is the major NAD+ consuming activity and plays a key role in maintaining genomic integrity. Scope of review: In this review, we discuss how different organelles are linked through NAD+ metabolism and how PARP1 activation in the nucleus can impact the function of distant organelles. We discuss how differentiated cells tame PARP1 function by upregulating an endogenous inhibitor, the histone variant macroH2A1.1. Major conclusions: The presence of macroH2A1.1, particularly in differentiated cells, raises the threshold for the activation of PARP1 with consequences for DNA repair, gene transcription, and NAD+ homeostasis. Research in the Ladurner and Buschbeck labs was supported by the following grants: the Deutsche Forschungsgemeinschaft SFB 646 and SFB 1064 collaborative research centers (to AGL); the Deutsche Forschungsgemeinschaft CIPSM and SyNergy research excellence clusters (to AGL); the Bavarian BioSysNet Program (to AGL); the ERA-NET Neuron project Food4Thought funded by the Bundesministerium für Bildung und Forschung (to AGL); the Marie Skłodowska Curie Training network “ChroMe” H2020-MSCA-ITN-2015-675,610 (to MB and AGL); MINECO RTI2018-094005-B-I00 (to MB); MINECO-ISCIII PIE16/00011 (to MB); the Deutsche José Carreras Leukaemie Stiftung DJCLS 14R/2018 (to MB); AGAUR 2017-SGR-305 (to MB); and Fundació La Marató de TV3 257/C/2019 (to MB). Research at the IJC is supported by the La Caixa Foundation, the Fundació Internacional Josep Carreras, Celgene Spain, and the CERCA Program/Generalitat de Catalunya.

Mammalian phosphatases of the haloacid dehalogenase (HAD) superfamily have emerged as important regulators of physiology and disease. Many of these enzymes are stable homodimers; however, the role of their dimerization is largely unknown. Here, we explore the function of the obligatory homodimerization of chronophin, a mammalian HAD phosphatase known to dephosphorylate pyridoxal 5'-phosphate (PLP) and serine/threonine-phosphorylated proteins. The exchange of two residues in the murine chronophin homodimerization interface (chronophin(A194K,A195K)) yields a constitutive monomer both in vitro and in cells. The catalytic activity of monomeric chronophin toward PLP is strongly impaired. X-ray crystallographic studies of chronophin(A194K,A195K) revealed that dimer formation is essential for an intermolecular arginine-arginine-tryptophan stacking interaction that positions a critical histidine residue in the substrate specificity loop of chronophin for PLP coordination. Analysis of all available crystal structures of HAD hydrolases that are grouped together with chronophin in the C2a-type structural subfamily uncovered a highly conserved mode of dimerization that results in intermolecular contacts involving the substrate specificity loop. Our results explain how the dimerization of HAD hydrolases contributes to their catalytic efficiency and substrate specificity.

DNA damage triggers chromatin remodeling by mechanisms that are poorly understood. The oncogene and chromatin remodeler ALC1/CHD1L massively decompacts chromatin in vivo yet is inactive prior to DNA-damage-mediated PARP1 induction. We show that the interaction of the ALC1 macrodomain with the ATPase module mediates auto-inhibition. PARP1 activation suppresses this inhibitory interaction. Crucially, release from auto-inhibition requires a poly-ADP-ribose (PAR) binding macrodomain. We identify tri-ADP-ribose as a potent PAR-mimic and synthetic allosteric effector that abrogates ATPase-macrodomain interactions, promotes an ungated conformation, and activates the remodeler’s ATPase. ALC1 fragments lacking the regulatory macrodomain relax chromatin in vivo without requiring PARP1 activation. Further, the ATPase restricts the macrodomain’s interaction with PARP1 under non-DNA damage conditions. Somatic cancer mutants disrupt ALC1’s auto-inhibition and activate chromatin remodeling. Our data show that the NAD+-metabolite and nucleic acid PAR triggers ALC1 to drive chromatin relaxation. Modular allostery in this oncogene tightly controls its robust, DNA-damage-dependent activation.

A set of phosphonic acid derivatives ( 1 – 4 ) of pyridoxal 5′-phosphate (PLP) was synthesized and characterized biochemically using purified murine pyridoxal phosphatase (PDXP), also known as chronophin. The most promising compound 1 displayed primarily competitive PDXP inhibitory activity with an IC 50 value of 79 μM, which was in the range of the K m of the physiological substrate PLP. We also report the X-ray crystal structure of PDXP bound to compound 3 , which we solved to 2.75 A resolution (PDB code 5AES). The co-crystal structure proves that compound 3 binds in the same orientation as PLP, and confirms the mode of inhibition to be competitive. Thus, we identify compound 1 as a PDXP phosphatase inhibitor. Our results suggest a strategy to design new, potent and selective PDXP inhibitors, which may be useful to increase the sensitivity of tumor cells to treatment with cytotoxic agents.

Mammalian haloacid dehalogenase (HAD)-type phosphatases are an emerging family of phosphatases with important functions in physiology and disease, yet little is known about the basis of their substrate specificity. Here, we characterize a previously unexplored HAD family member (gene annotation, phosphoglycolate phosphatase), which we termed AUM, for aspartate-based, ubiquitous, Mg(2+)-dependent phosphatase. AUM is a tyrosine-specific paralog of the serine/threonine-specific protein and pyridoxal 5'-phosphate-directed HAD phosphatase chronophin. Comparative evolutionary and biochemical analyses reveal that a single, differently conserved residue in the cap domain of either AUM or chronophin is crucial for phosphatase specificity. We have solved the x-ray crystal structure of the AUM cap fused to the catalytic core of chronophin to 2.65 Å resolution and present a detailed view of the catalytic clefts of AUM and chronophin that explains their substrate preferences. Our findings identify a small number of cap domain residues that encode the different substrate specificities of AUM and chronophin.