PARPs and ADP-ribosylation: Deciphering the complexity with molecular tools.

PARPs catalyze ADP-ribosylation-a post-translational modification that plays crucial roles in biological processes, including DNA repair, transcription, immune regulation, and condensate formation. ADP-ribosylation can be added to a wide range of amino acids with varying lengths and chemical structures, making it a complex and diverse modification. Despite this complexity, significant progress has been made in developing chemical biology methods to analyze ADP-ribosylated molecules and their binding proteins on a proteome-wide scale. Additionally, high-throughput assays have been developed to measure the activity of enzymes that add or remove ADP-ribosylation, leading to the development of inhibitors and new avenues for therapy. Real-time monitoring of ADP-ribosylation dynamics can be achieved using genetically encoded reporters, and next-generation detection reagents have improved the precision of immunoassays for specific forms of ADP-ribosylation. Further development and refinement of these tools will continue to advance our understanding of the functions and mechanisms of ADP-ribosylation in health and disease.

Defective ALC1 nucleosome remodeling confers PARPi sensitization and synthetic lethality with HRD.

Chromatin is a barrier to efficient DNA repair, as it hinders access and processing of certain DNA lesions. ALC1/CHD1L is a nucleosome-remodeling enzyme that responds to DNA damage, but its precise function in DNA repair remains unknown. Here we report that loss of ALC1 confers sensitivity to PARP inhibitors, methyl-methanesulfonate, and uracil misincorporation, which reflects the need to remodel nucleosomes following base excision by DNA glycosylases but prior to handover to APEX1. Using CRISPR screens, we establish that ALC1 loss is synthetic lethal with homologous recombination deficiency (HRD), which we attribute to chromosome instability caused by unrepaired DNA gaps at replication forks. In the absence of ALC1 or APEX1, incomplete processing of BER intermediates results in post-replicative DNA gaps and a critical dependence on HR for repair. Hence, targeting ALC1 alone or as a PARP inhibitor sensitizer could be employed to augment existing therapeutic strategies for HRD cancers.

PARPs and ADP-ribosylation: 60 years on.

Work on PARPs-a family of enzymes that catalyze ADP-ribosylation, a posttranslational modification of proteins-has resulted in major advances and reached important milestones. The past decade has seen new discoveries in areas well beyond the historical focus on DNA repair, which are having impacts on the understanding and treatment of human disease. This special focus section of Genes & Development includes seven reviews that highlight these discoveries and point the way forward for future advances in the field.

Chemical genetic methodologies for identifying protein substrates of PARPs.

Poly-ADP-ribose-polymerases (PARPs) are a family of 17 enzymes that regulate a diverse range of cellular processes in mammalian cells. PARPs catalyze the transfer of ADP-ribose from NAD+ to target molecules, most prominently amino acids on protein substrates, in a process known as ADP-ribosylation. Identifying the direct protein substrates of individual PARP family members is an essential first step for elucidating the mechanism by which PARPs regulate a particular pathway in cells. Two distinct chemical genetic (CG) strategies have been developed for identifying the direct protein substrates of individual PARP family members. In this review, we discuss the design principles behind these two strategies and how target identification has provided novel insight into the cellular function of individual PARPs and PARP-mediated ADP-ribosylation.

Structure-specific nucleases: role in Okazaki fragment maturation.

Proper function of structure-specific nucleases is key for faithful Okazaki fragment maturation (OFM) process completion. Deregulation of such nucleases leads to aberrant OFM and causes a spectrum of mutations, some of which may confer survival outcomes under specific stresses and serve as attractive targets for therapeutic intervention in human cancers.

Effects of poly (ADP-ribose) polymerase 1 (PARP1) on silk proteins in the silkworm, Bombyx mori.

Animal silk is economically important, while silk secretion is a complex and subtle mechanism regulated by many genes. We identified the poly (ADP-ribose) polymerase (PARP1) gene of the silkworm and successfully cloned its coding sequence (CDS) sequence. Using clustered regularly interspaced short palindromic repeat (CRISPR/Cas9) technology, we screened single guide RNA (sgRNA) with high knockout efficiency by cellular experiments and obtained PARP1 mutants by knocking out the PARP1 gene of the silkworm at the individual level. We found that the mutants mainly exhibited phenotypes such as smaller cocoon size and reduced cocoon shell rate than the wild type. We also detected the expression of silk protein genes in the mutant by quantitative real-time PCR (qPCR) and found that the expression of some silk protein genes was slightly down-regulated. Meanwhile, together with the results of transcriptomic analysis, we hypothesized that PARP1 may affect the synthesis of silk proteins, resulting in their failure to function properly. Our study may provide an important reference for future in-depth refinement of the molecular mechanism of silk protein expression in silk-producing animals, as well as a potential idea for future development of molecular breeding lines of silkworms to improve silk production.

Fueling genome maintenance: On the versatile roles of NAD+ in preserving DNA integrity.

NAD+ is a versatile biomolecule acting as a master regulator and substrate in various cellular processes, including redox regulation, metabolism, and various signaling pathways. In this article, we concisely and critically review the role of NAD+ in mechanisms promoting genome maintenance. Numerous NAD+-dependent reactions are involved in the preservation of genome stability, the cellular DNA damage response, and other pathways regulating nucleic acid metabolism, such as gene expression and cell proliferation pathways. Of note, NAD+ serves as a substrate to ADP-ribosyltransferases, sirtuins, and potentially also eukaryotic DNA ligases, all of which regulate various aspects of DNA integrity, damage repair, and gene expression. Finally, we critically analyze recent developments in the field as well as discuss challenges associated with therapeutic actions intended to raise NAD+ levels.

The Role of NAD+, SIRTs Interactions in Stimulating and Counteracting Carcinogenesis.

The World Health Organization has identified oncological diseases as one of the most serious health concerns of the current century. Current research on oncogenesis is focused on the molecular mechanisms of energy-biochemical reprogramming in cancer cell metabolism, including processes contributing to the Warburg effect and the pro-oncogenic and anti-oncogenic roles of sirtuins (SIRTs) and poly-(ADP-ribose) polymerases (PARPs). However, a clear understanding of the interaction between NAD+, SIRTs in cancer development, as well as their effects on carcinogenesis, has not been established, and literature data vary greatly. This work aims to provide a summary and structure of the available information on NAD+, SIRTs interactions in both stimulating and countering carcinogenesis, and to discuss potential approaches for pharmacological modulation of these interactions to achieve an anticancer effect.

Privileged Scaffolds for Potent and Specific Inhibitors of Mono-ADP-Ribosylating PARPs.

The identification of new targets to address unmet medical needs, better in a personalized way, is an urgent necessity. The introduction of PARP1 inhibitors into therapy, almost ten years ago, has represented a step forward this need being an innovate cancer treatment through a precision medicine approach. The PARP family consists of 17 members of which PARP1 that works by poly-ADP ribosylating the substrate is the sole enzyme so far exploited as therapeutic target. Most of the other members are mono-ADP-ribosylating (mono-ARTs) enzymes, and recent studies have deciphered their pathophysiological roles which appear to be very extensive with various potential therapeutic applications. In parallel, a handful of mono-ARTs inhibitors emerged that have been collected in a perspective on 2022. After that, additional very interesting compounds were identified highlighting the hot-topic nature of this research field and prompting an update. From the present review, where we have reported only mono-ARTs inhibitors endowed with the appropriate profile of pharmacological tools or drug candidate, four privileged scaffolds clearly stood out that constitute the basis for further drug discovery campaigns.

NAD+, Sirtuins and PARPs: enhancing oocyte developmental competence.

Oocyte quality is the limiting factor in female fertility. It is well known that maternal nutrition plays a role in reproductive function, and manipulating nutrition to improve fertility in livestock has been common practice in the past, particularly with respect to negative energy balance in cattle. A deficiency in nicotinamide adenine dinucleotide (NAD+) production has been associated with increased incidences of miscarriage and congenital defects in humans and mice, while elevating NAD+ through dietary supplements in aged subjects improved oocyte quality and embryo development. NAD+ is consumed by Sirtuins and poly-ADP-ribose polymerases (PARPs) within the cell and thus need constant replenishment in order to maintain various cellular functions. Sirtuins and PARPs play important roles in oocyte maturation and embryo development, and their activation may prove beneficial to in vitro embryo production and livestock breeding programs. This review examines the roles of NAD+, Sirtuins and PARPs in aspects of fertility, providing insights into the potential use of NAD+-elevating treatments in livestock breeding and embryo production programs.

A Double-Edged Sword: The Two Faces of PARylation.

Poly ADP-ribosylation (PARylation) is a post-translational modification process. Following the discovery of PARP-1, numerous studies have demonstrated the role of PARylation in the DNA damage and repair responses for cellular stress and DNA damage. Originally, studies on PARylation were confined to PARP-1 activation in the DNA repair pathway. However, the interplay between PARylation and DNA repair suggests that PARylation is important for the efficiency and accuracy of DNA repair. PARylation has contradicting roles; however, recent evidence implicates its importance in inflammation, metabolism, and cell death. These differences might be dependent on specific cellular conditions or experimental models used, and suggest that PARylation may play two opposing roles in cellular homeostasis. Understanding the role of PARylation in cellular function is not only important for identifying novel therapeutic approaches; it is also essential for gaining insight into the mechanisms of unexplored diseases. In this review, we discuss recent reports on the role of PARylation in mediating diverse cellular functions and homeostasis, such as DNA repair, inflammation, metabolism, and cell death.

Brain tissue transcriptomic analysis of SIV-infected macaques identifies several altered metabolic pathways linked to neuropathogenesis and poly (ADP-ribose) polymerases (PARPs) as potential therapeutic targets.

Despite improvements in antiretroviral therapy, human immunodeficiency virus type 1 (HIV-1)-associated neurocognitive disorders (HAND) remain prevalent in subjects undergoing therapy. HAND significantly affects individuals' quality of life, as well as adherence to therapy, and, despite the increasing understanding of neuropathogenesis, no definitive diagnostic or prognostic marker has been identified. We investigated transcriptomic profiles in frontal cortex tissues of Simian immunodeficiency virus (SIV)-infected Rhesus macaques sacrificed at different stages of infection. Gene expression was compared among SIV-infected animals (n = 11), with or without CD8+ lymphocyte depletion, based on detectable (n = 6) or non-detectable (n = 5) presence of the virus in frontal cortex tissues. Significant enrichment in activation of monocyte and macrophage cellular pathways was found in animals with detectable brain infection, independently from CD8+ lymphocyte depletion. In addition, transcripts of four poly (ADP-ribose) polymerases (PARPs) were up-regulated in the frontal cortex, which was confirmed by real-time polymerase chain reaction. Our results shed light on involvement of PARPs in SIV infection of the brain and their role in SIV-associated neurodegenerative processes. Inhibition of PARPs may provide an effective novel therapeutic target for HIV-related neuropathology.

PGC-1α, Sirtuins and PARPs in Huntington's Disease and Other Neurodegenerative Conditions: NAD+ to Rule Them All.

In this review, we summarize the available published information on the neuroprotective effects of increasing nicotinamide adenine dinucleotide (NAD+) levels in Huntington's disease models. We discuss the rationale of potential therapeutic benefit of administering nicotinamide riboside (NR), a safe and effective NAD+ precursor. We discuss the agonistic effect on the Sirtuin1-PGC-1α-PPAR pathway as well as Sirtuin 3, which converge in improving mitochondrial function, decreasing ROS production and ameliorating bioenergetics deficits. Also, we discuss the potential synergistic effect of increasing NAD+ combined with PARPs inhibitors, as a clinical therapeutic option not only in HD, but other neurodegenerative conditions.

The role of ADP-ribosylation in regulating DNA interstrand crosslink repair.

ADP-ribosylation by ADP-ribosyltransferases (ARTs) has a well-established role in DNA strand break repair by promoting enrichment of repair factors at damage sites through ADP-ribose interaction domains. Here, we exploit the simple eukaryote Dictyostelium to uncover a role for ADP-ribosylation in regulating DNA interstrand crosslink repair and redundancy of this pathway with non-homologous end-joining (NHEJ). In silico searches were used to identify a protein that contains a permutated macrodomain (which we call aprataxin/APLF-and-PNKP-like protein; APL). Structural analysis reveals that this permutated macrodomain retains features associated with ADP-ribose interactions and that APL is capable of binding poly(ADP-ribose) through this macrodomain. APL is enriched in chromatin in response to cisplatin treatment, an agent that induces DNA interstrand crosslinks (ICLs). This is dependent on the macrodomain of APL and the ART Adprt2, indicating a role for ADP-ribosylation in the cellular response to cisplatin. Although adprt2- cells are sensitive to cisplatin, ADP-ribosylation is evident in these cells owing to redundant signalling by the double-strand break (DSB)-responsive ART Adprt1a, promoting NHEJ-mediated repair. These data implicate ADP-ribosylation in DNA ICL repair and identify that NHEJ can function to resolve this form of DNA damage in the absence of Adprt2.

PARPs in genome stability and signal transduction: implications for cancer therapy.

The poly(ADP-ribose) polymerase (PARP) superfamily of enzymes catalyses the ADP-ribosylation (ADPr) of target proteins by using nicotinamide adenine dinucleotide (NAD+) as a donor. ADPr reactions occur either in the form of attachment of a single ADP-ribose nucleotide unit on target proteins or in the form of ADP-ribose chains, with the latter called poly(ADP-ribosyl)ation. PARPs regulate many cellular processes, including the maintenance of genome stability and signal transduction. In this review, we focus on the PARP family members that possess the ability to modify proteins by poly(ADP-ribosyl)ation, namely PARP1, PARP2, Tankyrase-1, and Tankyrase-2. Here, we detail the cellular functions of PARP1 and PARP2 in the regulation of DNA damage response and describe the function of Tankyrases in Wnt-mediated signal transduction. Furthermore, we discuss how the understanding of these pathways has provided some major breakthroughs in the treatment of human cancer.

Nonhomologous end-joining promotes resistance to DNA damage in the absence of an ADP-ribosyltransferase that signals DNA single strand breaks.

ADP-ribosylation of proteins at DNA lesions by ADP-ribosyltransferases (ARTs) is an early response to DNA damage. The best defined role of ADP-ribosylation in the DNA damage response is in repair of single strand breaks (SSBs). Recently, we initiated a study of how ADP-ribosylation regulates DNA repair in Dictyostelium and found that two ARTs (Adprt1b and Adprt2) are required for tolerance of cells to SSBs, and a third ART (Adprt1a) promotes nonhomologous end-joining (NHEJ). Here we report that disruption of adprt2 results in accumulation of DNA damage throughout the cell cycle following exposure to agents that induce base damage and DNA SSBs. Although ADP-ribosylation is evident in adprt2(-) cells exposed to methylmethanesulfonate (MMS), disruption of adprt1a and adprt2 in combination abolishes this response and further sensitises cells to this agent, indicating that in the absence of Adprt2, Adprt1a signals MMS-induced DNA lesions to promote resistance of cells to DNA damage. As a consequence of defective signalling of SSBs by Adprt2, Adprt1a is required to assemble NHEJ factors in chromatin, and disruption of the NHEJ pathway in combination with adprt2 increases sensitivity of cells to MMS. Taken together, these data indicate overlapping functions of different ARTs in signalling DNA damage, and illustrate a critical requirement for NHEJ in maintaining cell viability in the absence of an effective SSB response.

Selective inhibition of PARP10 using a chemical genetics strategy.

The lack of inhibitors that are selective for individual poly-ADP-ribose polymerase (PARP) family members has limited our understanding of their roles in cells. Here, we describe a chemical genetics approach for generating selective inhibitors of an engineered variant of PARP10. We synthesized a series of C-7 substituted 3,4-dihydroisoquinolin-1(2H)-one (dq) analogues designed to selectively inhibit a mutant of PARP10 (LG-PARP10) that contains a unique pocket in its active site. A dq analogue containing a bromo at the C-7 position demonstrated a 10-fold selectivity for LG-PARP10 compared to its WT counterpart. This study provides a platform for the development of selective inhibitors of individual PARP family members that will be useful for decoding their cellular functions.

ADP-ribosylation, a multifaceted modification: Functions and mechanisms in aging and aging-related diseases.

Aging, a complex biological process, plays key roles the development of multiple disorders referred as aging-related diseases involving cardiovascular diseases, stroke, neurodegenerative diseases, cancers, lipid metabolism-related diseases. ADP-ribosylation is a reversible modification onto proteins and nucleic acids to alter their structures and/or functions. Growing evidence support the importance of ADP-ribosylation and ADP-ribosylation-associated enzymes in aging and age-related diseases. In this review, we summarized ADP-ribosylation-associated proteins including ADP-ribosyl transferases, the ADP-ribosyl hydrolyses and ADP-ribose binding domains. Furthermore, we outlined the latest knowledge about regulation of ADP-ribosylation in the pathogenesis and progression of main aging-related diseases, organism aging and cellular senescence, and we also speculated the underlying mechanisms to better disclose this novel molecular network. Moreover, we discussed current issues and provided an outlook for future research, aiming to revealing the unknown bio-properties of ADP-ribosylation, and establishing a novel therapeutic perspective in aging-related diseases and health aging via targeting ADP-ribosylation.

PARsylation-mediated ubiquitylation: lessons from rare hereditary disease Cherubism.

Modification of proteins by ADP-ribose (PARsylation) is catalyzed by the poly(ADP-ribose) polymerase (PARP) family of enzymes exemplified by PARP1, which controls chromatin organization and DNA repair. Additionally, PARsylation induces ubiquitylation and proteasomal degradation of its substrates because PARsylation creates a recognition site for E3-ubiquitin ligase. The steady-state levels of the adaptor protein SH3-domain binding protein 2 (3BP2) is negatively regulated by tankyrase (PARP5), which coordinates ubiquitylation of 3BP2 by the E3-ligase ring finger protein 146 (RNF146). 3BP2 missense mutations uncouple 3BP2 from tankyrase-mediated negative regulation and cause Cherubism, an autosomal dominant autoinflammatory disorder associated with craniofacial dysmorphia. In this review, we summarize the diverse biological processes, including bone dynamics, metabolism, and Toll-like receptor (TLR) signaling controlled by tankyrase-mediated PARsylation of 3BP2, and highlight the therapeutic potential of this pathway.

A Clickable NAD+ Analog-Based Assay of Poly(ADP-Ribosyl)ated Proteins.

Poly(ADP-ribosyl)lation (PARylation) is a posttranslational modification that plays an important role in a variety of biological processes in both animals and plants. Identification of PARylated substrates is the key to elucidating the regulatory mechanism of PARylation. Several approaches have been developed to identify PARylated substrates over the past decade; however, a reliable and efficient method is needed to demonstrate PARylated proteins. Here, we report a simple and sensitive assay of PARylated proteins using a clickable 6-alkyne-NAD+ analog. The 6-alkyne-NAD+ is incorporated into substrate proteins in the in vitro PARylation assay. The labeled proteins are covalently captured by disulfide azide agarose beads through copper-catalyzed azide-alkyne cycloaddition (CuAAC), cleaved under reducing conditions, and analyzed by immunoblotting. The covalent bonds between the PARylated proteins and azide beads allow high stringent washing to eliminate nonspecific binding. Furthermore, the disulfide linker permits efficient cleavage and recovery of highly enriched PARylated proteins. Therefore, this approach can detect proteins that undergo PARylation at very low levels.