Iso-ADP-Ribose Fluorescence Polarization Probe for the Screening of RNF146 WWE Domain Inhibitors.

Poly-ADP-ribosylation is an important protein post-translational modification with diverse biological consequences. After binding poly-ADP-ribose on axis inhibition protein 1 (AXIN1) through its WWE domain, RING finger protein 146 (RNF146) can ubiquitinate AXIN1 and promote its proteasomal degradation and thus the oncogenic WNT signaling. Therefore, inhibiting the RNF146 WWE domain is a potential antitumor strategy. However, due to a lack of suitable screening methods, no inhibitors for this domain have been reported. Here, we developed a fluorescence polarization (FP)-based competition assay for the screening of RNF146 WWE inhibitors. This assay relies on a fluorescently tagged iso-ADP-ribose tracer compound, TAMRA-isoADPr. We report the design and synthesis of this tracer compound and show that it is a high-affinity tracer for the RNF146 WWE domain. This provides a convenient assay and will facilitate the development of small-molecule inhibitors for the RNF146 WWE domain.

Structural and biochemical analysis of the PARP1-homology region of PARP4/vault PARP.

PARP4 is an ADP-ribosyltransferase that resides within the vault ribonucleoprotein organelle. Our knowledge of PARP4 structure and biochemistry is limited relative to other PARPs. PARP4 shares a region of homology with PARP1, an ADP-ribosyltransferase that produces poly(ADP-ribose) from NAD+ in response to binding DNA breaks. The PARP1-homology region of PARP4 includes a BRCT fold, a WGR domain, and the catalytic (CAT) domain. Here, we have determined X-ray structures of the PARP4 catalytic domain and performed biochemical analysis that together indicate an active site that is open to NAD+ interaction, in contrast to the closed conformation of the PARP1 catalytic domain that blocks access to substrate NAD+. We have also determined crystal structures of the minimal ADP-ribosyltransferase fold of PARP4 that illustrate active site alterations that restrict PARP4 to mono(ADP-ribose) rather than poly(ADP-ribose) modifications. We demonstrate that PARP4 interacts with vault RNA, and that the BRCT is primarily responsible for the interaction. However, the interaction does not lead to stimulation of mono(ADP-ribosylation) activity. The BRCT-WGR-CAT of PARP4 has lower activity than the CAT alone, suggesting that the BRCT and WGR domains regulate catalytic output. Our study provides first insights into PARP4 structure and regulation and expands understanding of PARP structural biochemistry.

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

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.

Switch-like compaction of poly(ADP-ribose) upon cation binding.

Poly(ADP-ribose) (PAR) is a homopolymer of adenosine diphosphate ribose that is added to proteins as a posttranslational modification to regulate numerous cellular processes. PAR also serves as a scaffold for protein binding in macromolecular complexes, including biomolecular condensates. It remains unclear how PAR achieves specific molecular recognition. Here, we use single-molecule fluorescence resonance energy transfer (smFRET) to evaluate PAR flexibility under different cation conditions. We demonstrate that, compared to RNA and DNA, PAR has a longer persistence length and undergoes a sharper transition from extended to compact states in physiologically relevant concentrations of various cations (Na+, Mg2+, Ca2+, and spermine4+). We show that the degree of PAR compaction depends on the concentration and valency of cations. Furthermore, the intrinsically disordered protein FUS also served as a macromolecular cation to compact PAR. Taken together, our study reveals the inherent stiffness of PAR molecules, which undergo switch-like compaction in response to cation binding. This study indicates that a cationic environment may drive recognition specificity of PAR.

Development and characterization of new tools for detecting poly(ADP-ribose) in vitro and in vivo.

ADP-ribosylation (ADPRylation) is a reversible post-translation modification resulting in the covalent attachment of ADP-ribose (ADPR) moieties on substrate proteins. Naturally occurring protein motifs and domains, including WWEs, PBZs, and macrodomains, act as 'readers' for protein-linked ADPR. Although recombinant, antibody-like ADPR detection reagents containing these readers have facilitated the detection of ADPR, they are limited in their ability to capture the dynamic nature of ADPRylation. Herein, we describe and characterize a set of poly(ADP-ribose) (PAR) Trackers (PAR-Ts)-optimized dimerization-dependent or split-protein reassembly PAR sensors in which a naturally occurring PAR binding domain, WWE, was fused to both halves of dimerization-dependent GFP (ddGFP) or split Nano Luciferase (NanoLuc), respectively. We demonstrate that these new tools allow the detection and quantification of PAR levels in extracts, living cells, and living tissues with greater sensitivity, as well as temporal and spatial precision. Importantly, these sensors detect changes in cellular ADPR levels in response to physiological cues (e.g., hormone-dependent induction of adipogenesis without DNA damage), as well as xenograft tumor tissues in living mice. Our results indicate that PAR Trackers have broad utility for detecting ADPR in many different experimental and biological systems.

Hierarchical chemical determination of amyloid polymorphs in neurodegenerative disease.

Amyloid aggregation, which disrupts protein homeostasis, is a common pathological event occurring in human neurodegenerative diseases (NDs). Numerous evidences have shown that the structural diversity, so-called polymorphism, is decisive to the amyloid pathology and is closely associated with the onset, progression, and phenotype of ND. But how could one protein form so many stable structures? Recently, atomic structural evidence has been rapidly mounting to depict the involvement of chemical modifications in the amyloid fibril formation. In this Perspective, we aim to present a hierarchical regulation of chemical modifications including covalent post-translational modifications (PTMs) and noncovalent cofactor binding in governing the polymorphic amyloid formation, based mainly on the latest α-synuclein and Tau fibril structures. We hope to emphasize the determinant role of chemical modifications in amyloid assembly and pathology and to evoke chemical biological approaches to lead the fundamental and therapeutic research on protein amyloid state and the associated NDs.

DELTEX2 C-terminal domain recognizes and recruits ADP-ribosylated proteins for ubiquitination.

Cross-talk between ubiquitination and ADP-ribosylation regulates spatiotemporal recruitment of key players in many signaling pathways. The DELTEX family ubiquitin ligases (DTX1 to DTX4 and DTX3L) are characterized by a RING domain followed by a C-terminal domain (DTC) of hitherto unknown function. Here, we use two label-free mass spectrometry techniques to investigate the interactome and ubiquitinated substrates of human DTX2 and identify a large proportion of proteins associated with the DNA damage repair pathway. We show that DTX2-catalyzed ubiquitination of these interacting proteins requires PARP1/2-mediated ADP-ribosylation and depends on the DTC domain. Using a combination of structural, biochemical, and cell-based techniques, we show that the DTX2 DTC domain harbors an ADP-ribose-binding pocket and recruits poly-ADP-ribose (PAR)-modified proteins for ubiquitination. This PAR-binding property of DTC domain is conserved across the DELTEX family E3s. These findings uncover a new ADP-ribose-binding domain that facilitates PAR-dependent ubiquitination.

PARP1 catalytic variants reveal branching and chain length-specific functions of poly(ADP-ribose) in cellular physiology and stress response.

Poly(ADP-ribosyl)ation regulates numerous cellular processes like genome maintenance and cell death, thus providing protective functions but also contributing to several pathological conditions. Poly(ADP-ribose) (PAR) molecules exhibit a remarkable heterogeneity in chain lengths and branching frequencies, but the biological significance of this is basically unknown. To unravel structure-specific functions of PAR, we used PARP1 mutants producing PAR of different qualities, i.e. short and hypobranched (PARP1\G972R), short and moderately hyperbranched (PARP1\Y986S), or strongly hyperbranched PAR (PARP1\Y986H). By reconstituting HeLa PARP1 knockout cells, we demonstrate that PARP1\G972R negatively affects cellular endpoints, such as viability, cell cycle progression and genotoxic stress resistance. In contrast, PARP1\Y986S elicits only mild effects, suggesting that PAR branching compensates for short polymer length. Interestingly, PARP1\Y986H exhibits moderate beneficial effects on cell physiology. Furthermore, different PARP1 mutants have distinct effects on molecular processes, such as gene expression and protein localization dynamics of PARP1 itself, and of its downstream factor XRCC1. Finally, the biological relevance of PAR branching is emphasized by the fact that branching frequencies vary considerably during different phases of the DNA damage-induced PARylation reaction and between different mouse tissues. Taken together, this study reveals that PAR branching and chain length essentially affect cellular functions, which further supports the notion of a 'PAR code'.

PARG has a robust endo-glycohydrolase activity that releases protein-free poly(ADP-ribose) chains.

Poly(ADP-ribosyl)ation (PARylation) regulates DNA damage response, chromatin structure, and cell-fate. Dynamic regulation of cellular PAR levels is crucial for the maintenance of genomic integrity and excessive cellular PAR activates a PAR-dependent cell death pathway. Thus, PAR serves as a cell-death signal; however, it has been debated how the protein-free PAR is generated. Here, we demonstrate that PAR glycohydrolases (PARGs) from mammals to bacteria have a robust endo-glycohydrolase activity, releasing protein-free PAR chains longer than three ADP-ribose units as early reaction products. Released PAR chains are transient and rapidly degraded to monomeric ADP-ribose, which is consistent with a short half-life of PAR during DNA damage responses. Computational simulations using a tri-ADP-ribose further support that PARG can efficiently bind to internal sites of PAR for the endo-glycosidic cleavage. Our collective results suggest PARG as a key player in producing protein-free PAR during DNA damage signaling and establish bacterial PARG as a useful tool to enrich short PAR chains that emerge as important reagents for biomedical research.

Modeling of the Enzyme-Substrate Complexes of Human Poly(ADP-Ribose) Polymerase 1.

Poly(ADP-ribose) polymerase 1 (PARP-1) is a key DNA repair enzyme and an important target in cancer treatment. Conventional methods of studying the reaction mechanism of PARP-1 have limitations because of the complex structure of PARP-1 substrates; however, the necessary data can be obtained by molecular modeling. In this work, a molecular dynamics model for the PARP-1 enzyme-substrate complex containing NAD+ molecule and the end of the poly(ADP-ribose) chain in the form of ADP molecule was obtained for the first time. Interactions with the active site residues have been characterized where Gly863, Lys903, Glu988 play a crucial role, and the SN1-like mechanism for the enzymatic ADP-ribosylation reaction has been proposed. Models of PARP-1 complexes with more sophisticated two-unit fragments of the growing polymer chain as well as competitive inhibitors 3-aminobenzamide and 7-methylguanine have been obtained by molecular docking.

Poly(ADP-ribose)-dependent ubiquitination and its clinical implications.

ADP-ribosylation-the addition of one or multiple ADP-ribose units onto proteins-is a therapeutically important post-translational modification implicated in cancer, neurodegeneration, and infectious diseases. The protein modification regulates a broad range of biological processes, including DNA repair, transcription, RNA metabolism, and the structural integrity of nonmembranous structures. The polymeric form of ADP-ribose, poly(ADP-ribose), was recently identified as a signal for triggering protein degradation through the ubiquitin-proteasome system. Using informatics analyses, we found that these ubiquitinated substrates tend to be low abundance proteins, which may serve as rate-limiting factors within signaling networks or metabolic processes. In this review, we summarize the current literature on poly(ADP-ribose)-dependent ubiquitination (PARdU) regarding its biological mechanisms, substrates, and relevance to diseases.

Structural analyses of NudT16-ADP-ribose complexes direct rational design of mutants with improved processing of poly(ADP-ribosyl)ated proteins.

ADP-ribosylation is a post-translational modification that occurs on chemically diverse amino acids, including aspartate, glutamate, lysine, arginine, serine and cysteine on proteins and is mediated by ADP-ribosyltransferases, including a subset commonly known as poly(ADP-ribose) polymerases. ADP-ribose can be conjugated to proteins singly as a monomer or in polymeric chains as poly(ADP-ribose). While ADP-ribosylation can be reversed by ADP-ribosylhydrolases, this protein modification can also be processed to phosphoribosylation by enzymes possessing phosphodiesterase activity, such as snake venom phosphodiesterase, mammalian ectonucleotide pyrophosphatase/phosphodiesterase 1, Escherichia coli RppH, Legionella pneumophila Sde and Homo sapiens NudT16 (HsNudT16). Our studies here sought to utilize X-ray crystallographic structures of HsNudT16 in complex with monomeric and dimeric ADP-ribose in identifying the active site for binding and processing free and protein-conjugated ADP-ribose into phosphoribose forms. These structural data guide rational design of mutants that widen the active site to better accommodate protein-conjugated ADP-ribose. We identified that several HsNudT16 mutants (Δ17, F36A, and F61S) have reduced activity for free ADP-ribose, similar processing ability against protein-conjugated mono(ADP-ribose), but improved catalytic efficiency for protein-conjugated poly(ADP-ribose). These HsNudT16 variants may, therefore, provide a novel tool to investigate different forms of ADP-ribose.

Structural and biochemical evidence supporting poly ADP-ribosylation in the bacterium Deinococcus radiodurans.

Poly-ADP-ribosylation, a post-translational modification involved in various cellular processes, is well characterized in eukaryotes but thought to be devoid in bacteria. Here, we solve crystal structures of ADP-ribose-bound poly(ADP-ribose)glycohydrolase from the radioresistant bacterium Deinococcus radiodurans (DrPARG), revealing a solvent-accessible 2'-hydroxy group of ADP-ribose, which suggests that DrPARG may possess endo-glycohydrolase activity toward poly-ADP-ribose (PAR). We confirm the existence of PAR in D. radiodurans and show that disruption of DrPARG expression causes accumulation of endogenous PAR and compromises recovery from UV radiation damage. Moreover, endogenous PAR levels in D. radiodurans are elevated after UV irradiation, indicating that PARylation may be involved in resistance to genotoxic stresses. These findings provide structural insights into a bacterial-type PARG and suggest the existence of a prokaryotic PARylation machinery that may be involved in stress responses.

Interactions of p53 with poly(ADP-ribose) and DNA induce distinct changes in protein structure as revealed by ATR-FTIR spectroscopy.

Due to multiple domains and in part intrinsically disordered regions, structural analyses of p53 remain a challenging task, particularly in complex with DNA and other macromolecules. Here, we applied a novel attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopic approach to investigate changes in secondary structure of full-length p53 induced by non-covalent interactions with DNA and poly(ADP-ribose) (PAR). To validate our approach, we confirmed a positive regulatory function of p53's C-terminal domain (CTD) with regard to sequence-specific DNA binding and verified that the CTD mediates p53-PAR interaction. Further, we demonstrate that DNA and PAR interactions result in distinct structural changes of p53, indicating specific binding mechanisms via different domains. A time-dependent analysis of the interplay of DNA and PAR binding to p53 revealed that PAR represents p53's preferred binding partner, which efficiently controls p53-DNA interaction. Moreover, we provide infrared spectroscopic data on PAR pointing to the absence of regular secondary structural elements. Finally, temperature-induced melting experiments via CD spectroscopy show that DNA binding stabilizes the structure of p53, while PAR binding can shift the irreversible formation of insoluble p53 aggregates to higher temperatures. In conclusion, this study provides detailed insights into the dynamic interplay of p53 binding to DNA and PAR at a formerly inaccessible molecular level.

NR4A Nuclear Receptors Target Poly-ADP-Ribosylated DNA-PKcs Protein to Promote DNA Repair.

Although poly-ADP-ribosylation (PARylation) of DNA repair factors had been well documented, its role in the repair of DNA double-strand breaks (DSBs) is poorly understood. NR4A nuclear orphan receptors were previously linked to DSB repair; however, their function in the process remains elusive. Classically, NR4As function as transcription factors using a specialized tandem zinc-finger DNA-binding domain (DBD) for target gene induction. Here, we show that NR4A DBD is bi-functional and can bind poly-ADP-ribose (PAR) through a pocket localized in the second zinc finger. Separation-of-function mutants demonstrate that NR4A PAR binding, while dispensable for transcriptional activity, facilitates repair of radiation-induced DNA double-strand breaks in G1. Moreover, we define DNA-PKcs protein as a prominent target of ionizing radiation-induced PARylation. Mechanistically, NR4As function by directly targeting poly-ADP-ribosylated DNA-PKcs to facilitate its autophosphorylation-promoting DNA-PK kinase assembly at DNA lesions. Selective targeting of the PAR-binding pocket of NR4A presents an opportunity for cancer therapy.

The WD40 domain of FBXW7 is a poly(ADP-ribose)-binding domain that mediates the early DNA damage response.

FBXW7, a classic tumor suppressor, is a substrate recognition subunit of the Skp1-cullin-F-box (SCF) ubiquitin ligase that targets oncoproteins for ubiquitination and degradation. We recently found that FBXW7 is recruited to DNA damage sites to facilitate nonhomologous end-joining (NHEJ). The detailed underlying molecular mechanism, however, remains elusive. Here we report that the WD40 domain of FBXW7, which is responsible for substrate binding and frequently mutated in human cancers, binds to poly(ADP-ribose) (PAR) immediately following DNA damage and mediates rapid recruitment of FBXW7 to DNA damage sites, whereas ATM-mediated FBXW7 phosphorylation promotes its retention at DNA damage sites. Cancer-associated arginine mutations in the WD40 domain (R465H, R479Q and R505C) abolish both FBXW7 interaction with PAR and recruitment to DNA damage sites, causing inhibition of XRCC4 polyubiquitination and NHEJ. Furthermore, inhibition or silencing of poly(ADP-ribose) polymerase 1 (PARP1) inhibits PAR-mediated recruitment of FBXW7 to the DNA damage sites. Taken together, our study demonstrates that the WD40 domain of FBXW7 is a novel PAR-binding motif that facilitates early recruitment of FBXW7 to DNA damage sites for subsequent NHEJ repair. Abrogation of this ability seen in cancer-derived FBXW7 mutations provides a molecular mechanism for defective DNA repair, eventually leading to genome instability.

Synthesis of disaccharide nucleoside analogues as potential poly(ADP-ribose) polymerase-1 inhibitors.

Poly(ADP-ribose) polymerase-1 (PARP-1) is an important target in cancer therapy. We present the synthesis of novel disaccharide nucleoside analogues that resemble the central motif of poly(ADP-ribose) and test their inhibitory effects on human PARP-1. Some compounds show inhibition of enzymatic activity in vitro and thus might be interesting for further investigations.

Biochemical and Biophysical Assays of PAR-WWE Domain Interactions and Production of iso-ADPr for PAR-Binding Analysis.

The poly(ADP-ribose) polymerase (PARP) family of proteins utilize NAD+ as the substrate to modify protein acceptors with either mono(ADP-ribose) (MAR) or poly(ADP-ribose) (PAR). MAR and PAR have been shown to regulate distinct cellular processes. Iso-ADP-ribose (iso-ADPr) is the smallest internal PAR structural unit containing the characteristic ribose-ribose glycosidic bond formed during poly(ADP-ribosyl)ation. The WWE domain of RNF146 specifically recognizes the iso-ADPr moiety in PAR but does not interact with MAR. This provides a way to distinguish PAR from MAR modification and to isolate PARylated proteins. Iso-ADPr can be used to detect the PAR-specific binding properties of interested proteins. Here we describe the detailed method to generate and purify iso-ADPr and its use in PAR-binding analysis through isothermal titration calorimetry (ITC) analysis.

Generating Protein-Linked and Protein-Free Mono-, Oligo-, and Poly(ADP-Ribose) In Vitro.

ADP-ribosylation is a covalent posttranslational modification of proteins that is catalyzed by various types of ADP-ribosyltransferase (ART) enzymes, including members of the poly(ADP-ribose) polymerase (PARP) family. ADP-ribose (ADPR) modifications can occur as mono(ADP-ribosyl)ation, oligo(ADP-ribosyl)ation, or poly(ADP-ribosyl)ation, depending on the particular ART enzyme catalyzing the reaction, as well as the specific reaction conditions. Understanding the biology of ADP-ribosylation requires facile and robust means of generating and detecting the modification in all of its forms. Here we describe how to generate protein-linked mono(ADP-ribose), oligo(ADP-ribose), and poly(ADP-ribose) (MAR, OAR, and PAR, respectively) in vitro as an automodification of PARPs 1 or 3. First, epitope-tagged PARP-1 (a PARP polyenzyme) and PARP-3 (a PARP monoenzyme) are expressed individually in insect cells using baculovirus expression vectors, and purified using immunoaffinity chromatography. Second, the purified recombinant PARPs are incubated individually in the presence of different concentrations of NAD+ (as a donor of ADPR groups) and sheared DNA (to activate their catalytic activities) resulting in various forms of auto-ADP-ribosylation. Third, the products are confirmed using ADPR detection reagents that can distinguish among MAR, OAR, and PAR. Finally, if desired, the OAR and PAR can be deproteinized. The protein-linked and free MAR, OAR, and PAR generated in these reactions can be used as standards, substrates, or binding partners in a variety of ADPR-related assays.

ADP-Ribosyl-Acceptor Hydrolase Activities Catalyzed by the ARH Family of Proteins.

The ARH family of ADP-ribosyl-acceptor hydrolases is composed of three 39-kDa proteins (ARH1, 2, and 3), which hydrolyze specific ADP-ribosylated substrates. ARH1 hydrolyzes mono(ADP-ribosyl)ated arginine, which results from actions of cholera toxin and other nicotinamide adenine dinucleotide (NAD+):arginine ADP-ribosyl-transferases, while ARH3 hydrolyzes poly(ADP-ribose) and O-acetyl-ADP-ribose, resulting from the action of poly(ADP-ribose) polymerases and sirtuins, respectively. ARH2 has not been reported to have enzymatic activity, because of differences in the catalytic domain. Thus, the substrate specificities of ARH1 and ARH3 proteins result in unique cellular functions. In this chapter, we introduce several methods to monitor the activities of the ARH family members.