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Nucleotide-binding domain and leucine-rich repeat pyrin-domain containing protein 1 (NLRP1) is an inflammasome sensor that mediates the activation of caspase-1 to induce cytokine maturation and pyroptosis.sup.1-4. Gain-of-function mutations of NLRP1 cause severe inflammatory diseases of the skin.sup.4-6. NLRP1 contains a function-to-find domain that auto-proteolyses into noncovalently associated subdomains.sup.7-9, and proteasomal degradation of the repressive N-terminal fragment of NLRP1 releases its inflammatory C-terminal fragment (NLRP1 CT).sup.10,11. Cytosolic dipeptidyl peptidases 8 and 9 (hereafter, DPP8/DPP9) both interact with NLRP1, and small-molecule inhibitors of DPP8/DPP9 activate NLRP1 by mechanisms that are currently unclear.sup.10,12-14. Here we report cryo-electron microscopy structures of the human NLRP1-DPP9 complex alone and with Val-boroPro (VbP), an inhibitor of DPP8/DPP9. The structures reveal a ternary complex that comprises DPP9, full-length NLRP1 and the NLRPT CT. The binding of the NLRP1 CT to DPP9 requires full-length NLRP1, which suggests that NLRP1 activation is regulated by the ratio of NLRP1 CT to full-length NLRP1. Activation of the inflammasome by ectopic expression of the NLRP1 CT is consistently rescued by co-expression of autoproteolysis-deficient full-length NLRP1. The N terminus of the NLRP1 CT inserts into the DPP9 active site, and VbP disrupts this interaction. Thus, VbP weakens the NLRP1-DPP9 interaction and accelerates degradation of the N-terminal fragment.sup.10 to induce inflammasome activation. Overall, these data demonstrate that DPP9 quenches low levels of NLRP1 CT and thus serves as a checkpoint for activation of the NLRP1 inflammasome. Structures of NLRP1-DPP9 alone and with a small-molecule inhibitor of DPP9 reveal the mechanisms through which NLRP1 is regulated, providing insights into the role of this complex in inflammasome regulation., Author(s): L. Robert Hollingsworth [sup.1] [sup.2] [sup.3] , Humayun Sharif [sup.1] [sup.2] , Andrew R. Griswold [sup.4] [sup.5] , Pietro Fontana [sup.1] [sup.2] , Julian Mintseris [sup.6] , Kevin B. [...]

Author(s): Marcin J. Suskiewicz [sup.1] , Florian Zobel [sup.1] , Tom E. H. Ogden [sup.2] , Pietro Fontana [sup.1] , Antonio Ariza [sup.1] , Ji-Chun Yang [sup.2] , Kang Zhu [...], The anti-cancer drug target poly(ADP-ribose) polymerase 1 (PARP1) and its close homologue, PARP2, are early responders to DNA damage in human cells.sup.1,2. After binding to genomic lesions, these enzymes use NAD.sup.+ to modify numerous proteins with mono- and poly(ADP-ribose) signals that are important for the subsequent decompaction of chromatin and the recruitment of repair factors.sup.3,4. These post-translational modifications are predominantly serine-linked and require the accessory factor HPF1, which is specific for the DNA damage response and switches the amino acid specificity of PARP1 and PARP2 from aspartate or glutamate to serine residues.sup.5-10. Here we report a co-structure of HPF1 bound to the catalytic domain of PARP2 that, in combination with NMR and biochemical data, reveals a composite active site formed by residues from HPF1 and PARP1 or PARP2 . The assembly of this catalytic centre is essential for the addition of ADP-ribose moieties after DNA damage in human cells. In response to DNA damage and occupancy of the NAD.sup.+-binding site, the interaction of HPF1 with PARP1 or PARP2 is enhanced by allosteric networks that operate within the PARP proteins, providing an additional level of regulation in the induction of the DNA damage response. As HPF1 forms a joint active site with PARP1 or PARP2, our data implicate HPF1 as an important determinant of the response to clinical PARP inhibitors. Assembly of a catalytic centre formed by HPF1 bound to PARP1 or PARP2 is essential for protein ADP-ribosylation after DNA damage in human cells.

Chemical modifications of RNA have key roles in many biological processes1–3. N7-methylguanosine (m7G) is required for integrity and stability of a large subset of tRNAs4–7. The methyltransferase 1–WD repeat-containing protein 4 (METTL1–WDR4) complex is the methyltransferase that modifies G46 in the variable loop of certain tRNAs, and its dysregulation drives tumorigenesis in numerous cancer types8–14. Mutations in WDR4 cause human developmental phenotypes including microcephaly15–17. How METTL1–WDR4 modifies tRNA substrates and is regulated remains elusive18. Here we show, through structural, biochemical and cellular studies of human METTL1–WDR4, that WDR4 serves as a scaffold for METTL1 and the tRNA T-arm. Upon tRNA binding, the αC region of METTL1 transforms into a helix, which together with the α6 helix secures both ends of the tRNA variable loop. Unexpectedly, we find that the predicted disordered N-terminal region of METTL1 is part of the catalytic pocket and essential for methyltransferase activity. Furthermore, we reveal that S27 phosphorylation in the METTL1 N-terminal region inhibits methyltransferase activity by locally disrupting the catalytic centre. Our results provide a molecular understanding of tRNA substrate recognition and phosphorylation-mediated regulation of METTL1–WDR4, and reveal the presumed disordered N-terminal region of METTL1 as a nexus of methyltransferase activity.Structures of the human METTL1–WDR4 complex are revealed, providing molecular insights into substrate recognition, modification and catalytic regulation by the N7-methylguanosine methyltransferase complex. [ABSTRACT FROM AUTHOR]