ADP-ribosylation, a modification of proteins, nucleic acids and metabolites, confers broad functions, including roles in stress responses elicited for example by DNA damage and viral infection and is involved in intra-and extracellular signaling, chromatin and transcriptional regulation, protein biosynthesis and cell death. ADP-ribosylation is catalyzed by ADPribosyltransferases, which transfer ADP-ribose from NAD + onto substrates. The modification, which occurs as mono-or poly-ADP-ribosylation, is reversible due to the action of different ADPribosylhydrolases. Importantly, inhibitors of ADP-ribosyltransferases are approved or are being developed for clinical use. Moreover, ADP-ribosylhydrolases are being assessed as therapeutic targets, foremost as anti-viral drugs and for oncological indications. Due to the development of novel reagents and major technological advances that allow the study of ADP-ribosylation in unprecedented detail, an increasing number of cellular processes and pathways are being
Several cellular signaling pathways are regulated by ADP-ribosylation, a posttranslational modification catalyzed by members of the ARTD superfamily. Tankyrases are distinguishable from the rest of this family by their unique domain organization, notably the sterile alpha motif responsible for oligomerization and ankyrin repeats mediating protein-protein interactions. Tankyrases are involved in various cellular functions, such as telomere homeostasis, Wnt/β-catenin signaling, glucose metabolism, and cell cycle progression. In these processes, Tankyrases regulate the interactions and stability of target proteins by poly (ADP-ribosyl)ation. Modified proteins are subsequently recognized by the E3 ubiquitin ligase RNF146, poly-ubiquitinated and predominantly guided to 26S proteasomal degradation. Several small molecule inhibitors have been described for Tankyrases; they compete with the co-substrate NAD+ for binding to the ARTD catalytic domain. The recent, highly potent and selective inhibitors possess several properties of lead compounds and can be used for proof-of-concept studies in cancer and other Tankyrase linked diseases.
DEXD/H-box RNA helicases couple ATP hydrolysis to RNA remodeling by an unknown mechanism. We used x-ray crystallography and biochemical analysis of the human DEXD/H-box protein DDX19 to investigate its regulatory mechanism. The crystal structures of DDX19, in its RNA-bound prehydrolysis and free posthydrolysis state, reveal an ␣-helix that inserts between the conserved domains of the free protein to negatively regulate ATPase activity. This finding was corroborated by biochemical data that confirm an autoregulatory function of the N-terminal region of the protein. This is the first study describing crystal structures of a DEXD/H-box protein in its open and closed cleft conformations.RNA helicase activity is involved in all aspects of RNA metabolism, including transcription, pre-mRNA splicing, ribosome biogenesis, nuclear export, translation initiation and termination, RNA degradation, viral replication, and viral RNA detection. The DEXD/H-box RNA helicases couple hydrolysis of ATP to cycles of RNA binding and release that typically result in non-processive RNA duplex unwinding (1) or disruption of RNP 3 complexes (2, 3). These proteins interact in a non-sequence-specific manner with the phosphoribose backbone of single-stranded RNA. DEXD/H-box RNA helicases contain two ␣/-RecA-like domains that both feature conserved sequence motifs involved in RNA binding and ATP hydrolysis (4, 5). Accessory proteins are involved in the regulation of RNA binding and ATPase activities, although no general mechanism has been demonstrated.The DDX19 member of the DEXD/H-box RNA helicase family performs an essential function in mRNA nuclear export by remodeling RNP particles during passage of mRNA through the nuclear pore complex (3, 6). Dbp5, the yeast orthologue of DDX19 (7,8), causes displacement of the RNP constituent, Mex67, thereby preventing re-entry of mRNA into the nucleus (9). Dbp5 is also involved in translation termination (10). A specific function has been assigned to the ADP-bound form of Dbp5, which displaces the RNA-binding protein Nab2, an event that is required for mRNA export (3). In vivo, Dbp5 is activated by the nuclear pore complex-associated protein, Gle1 (11,12). Crystal structures of DEXD/H-box proteins show two-lobed proteins with the nucleotide binding site located in the lower part of the cleft separating the conserved domains and the RNA binding site across the upper cleft opening (13-17). DEXD/Hbox helicases in general share little homology in their coding sequences upstream of the conserved domain-1. The N-terminal extension of DDX19, however, shares significant homology with that of DDX25/GRTH, a testis-specific protein that is essential for spermatogenesis (18), supporting a functional significance for this sequence. Herein, we present a crystal structure of human DDX19 that shows the ADP-bound protein with an ␣-helical segment of the N-terminal extension wedged between the core domains, preventing cleft closure. In the structure of the ADPNP-bound protein in complex with RNA, this ␣-helix has moved...
Tankyrase 1 and tankyrase 2 are poly(ADP-ribosyl)ases that are distinguishable from other members of the enzyme family by the structural features of the catalytic domain, and the presence of a sterile a-motif multimerization domain and an ankyrin repeat protein-interaction domain. Tankyrases are implicated in a multitude of cellular functions, including telomere homeostasis, mitotic spindle formation, vesicle transport linked to glucose metabolism, Wnt-b-catenin signaling, and viral replication. In these processes, tankyrases interact with target proteins, catalyze poly (ADP-ribosyl)ation, and regulate protein interactions and stability. The proposed roles of tankyrases in disease-relevant cellular processes have made them attractive drug targets. Recently, several inhibitors have been identified. The selectivity and potency of these small molecules can be rationalized by how they fit within the NAD + -binding groove of the catalytic domain. Some molecules bind to the nicotinamide subsite, such as generic diphtheria toxin-like ADP-ribosyltransferase inhibitors, whereas others bind to a distinct adenosine subsite that diverges from other diphtheria toxin-like ADP-ribosyltransferases and confers specificity. A highly potent dual-site inhibitor is also available. Within the last few years, tankyrase inhibitors have proved to be useful chemical probes and potential lead compounds, especially for specific cancers.
Tankyrases are poly(ADP-ribose) polymerases that have many cellular functions. They play pharmaceutically important roles, at least in telomere homeostasis and Wnt signaling, by covalently ADP-ribosylating target proteins and consequently regulating their functions. These features make tankyrases potential targets for treatment of cancer. We report here crystal structures of human tankyrase 2 catalytic fragment in complex with a byproduct, nicotinamide, and with selective inhibitors of tankyrases (IWR-1) and PARPs 1 and 2 (olaparib). Binding of these inhibitors to tankyrase 2 induces specific conformational changes. The crystal structures explain the selectivity of the inhibitors, reveal the flexibility of a substrate binding loop, and explain existing structure-activity relationship data. The first crystal structure of a PARP enzyme in complex with a potent inhibitor, IWR-1, that does not bind to the widely utilized nicotinamide-binding site makes the structure valuable for development of PARP inhibitors in general.
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