Abstract: Inhibitor of apoptosis proteins (IAPs) such as XIAP, cIAP1, and cIAP2 are upregulated in many cancer cells. Several compounds targeting IAPs and inducing cell death in cancer cells have been developed. Some of these are synthesized mimicking the N-terminal tetrapeptide sequence of Smac/DIABLO, the natural endogenous IAPs inhibitor. Starting from such conceptual design, we generated a library of 4-substituted azabicyclo[5.3.0]alkane Smac-mimetics. Here we report the crystal structure of the BIR3 domain from XIAP in complex with Smac037, a compound designed according to structural principles emerging from our previously analyzed XIAP BIR3/Smac-mimetic complexes. In parallel, we present an in silico docking analysis of three Smac-mimetics to the BIR3 domain of cIAP1, providing general considerations for the development of high affinity lead compounds targeting three members of the IAP family.

Abstract: The X-linked inhibitor of apoptosis protein (XIAP) is overexpressed in several malignant cells where it prevents apoptosis by binding to, and blocking, the activation of caspase-3, -7, and -9. Human XIAP (479 residues) is composed of three tandem-repeated baculoviral IAP repeat (BIR) domains (BIR1-3), and by a C-terminal RING domain. Smac-DIABLO [second mitochondria-derived activator of caspases (Smac)-direct IAP binding protein with low pI (DIABLO)], the natural antagonist of XIAP, binds through its N-terminal sequence AVPI to the same surface groove, in the BIR domains, that binds caspases. Synthetic compounds mimicking such tetrapeptide motif effectively block the interaction between IAP and active caspases, thus triggering apoptosis. Peptidomimetics based on an azabicyclo[x.y.0]alkane scaffolds, have been shown to bind the BIR3 domain of XIAP with micromolar to nanomolar affinities, thus presenting attractive features for drug lead optimization. Here we report a study on three newly synthesized Smac mimetics, which have been characterized in their complexes with XIAP BIR3 domain through X-ray crystallography and molecular modelling/docking simulations. Based on analysis of the crystal structures, we show that specific substitutions at the 4-position of the azabicyclo[5.3.0]alkane scaffold results in sizeable effects on the peptidomimetic-BIR3 domain affinity. By means of functional, biophysical and simulative approaches we also propose that the same Smac mimetics can bind XIAP BIR2 domain at a location structurally related to the BIR3 domain AVPI binding groove. Details of the XIAP-Smac mimetic recognition principles highlighted by this study are discussed in light of the drug-like profile of the three (potentially proapoptotic) compounds developed that show improved performance in ADMET (adsorption, distribution, metabolism, excretion and toxicity) tests.

Abstract: REGgamma is a member of the 11S regulatory particle that activates the 20S proteasome. Studies in REGgamma deficient mice indicated an additional role for this protein in cell cycle regulation and proliferation control. In this paper we demonstrate that REGgamma protein is equally expressed throughout the cell cycle, but undergoes a distinctive subcellular localization at mitosis. Thus, while in interphase cells REGgamma is nuclear, in telophase cells it localizes on chromosomes, suggesting a role in mitotic progression. Furthermore, we found that REGgamma overexpression weakens the mitotic arrest induced by spindle damage, allowing premature exit from mitosis, whereas REGgamma depletion has the opposite effect, thus reflecting a new REGgamma function, unrelated to its role as proteasome activator. Additionally, we found that primary cells from REGgamma-/- mice and human fibroblasts with depleted expression of REGgamma or overexpressing a dominant negative mutant unable to activate the 20S proteasome, demonstrated a marked aneuploidy (chromosomal gains and losses), supernumerary centrosomes and multipolar spindles. These findings thus underscore a previously uncharacterized function of REGgamma in centrosome and chromosomal stability maintenance.

Abstract: Che-1 is a RNA polymerase II-binding protein involved in the transcription of E2F target genes and induction of cell proliferation. Here we show that Che-1 contributes to DNA damage response and that its depletion sensitizes cells to anticancer agents. The checkpoint kinases ATM/ATR and Chk2 interact with Che-1 and promote its phosphorylation and accumulation in response to DNA damage. These Che-1 modifications induce a specific recruitment of Che-1 on the TP53 and p21 promoters. Interestingly, it has a profound effect on the basal expression of p53, which is preserved following DNA damage. Notably, Che-1 contributes to the maintenance of the G2/M checkpoint induced by DNA damage. These findings identify a mechanism by which checkpoint kinases regulate responses to DNA damage.

Abstract: Artemis plays a crucial role in the hairpin-opening step of antigen receptor VDJ gene recombination in the presence of catalytic subunit of deoxyribonucleic acid (DNA)-dependent protein kinase (DNA-PKcs). A defect in Artemis causes human radiosensitive-severe combined immunodeficiency. Cells from Artemis-deficient patients and mice display increased chromosomal instability, but the precise function of this factor in the response to DNA damage remains to be elucidate. In this study, we show that Artemis is hyperphosphorylated in an Ataxia-telangiectasia-mutated (ATM)- and Nijmegen breakage syndrome 1 (Nbs1)-dependent manner in response to ionizing radiation (IR), and that S645 is an SQ/TQ site that contributes to retarded mobility of Artemis upon IR. The hyperphosphorylation of Artemis is markedly reduced in ATM- and Nbs1-null cells. Reintroduction of wild-type ATM or Nbs1 reconstituted Artemis hyperphosphorylation in ATM- or Nbs1-deficient cells, respectively. In support of this functional link, hyperphosphorylated Artemis was found to physically associate with the Mre11/Rad50/Nbs1 complex in an ATM-dependent manner in response to IR-induced DNA double strand breaks (DSB). Since deficiency of either DNA-Pkcs or ATM leads to defective repair of IR-induced DSB, our finding places Artemis at the signaling crossroads downstream of DNA-PKcs and ATM in IR-induced DSB repair.

Abstract: Chk2 is a nuclear protein kinase involved in the DNA damage-induced ataxia telangiectasia mutated-dependent checkpoint arrest at multiple cell cycle phases. Searching for Chk2-binding proteins by a yeast two-hybrid system, we identified a strong interaction with karyopherin-alpha2 (KPNA-2), a gene product involved in active nuclear import of proteins bearing a nuclear localization signal (NLS). This finding was confirmed by glutathione S-transferase pull-down and co-immunoprecipitation assays. Of the three predicted Chk2 NLSs, located at amino acids 179-182 (NLS-1), 240-256 (NLS-2), and 515-522 (NLS-3), only the latter mediated the interaction with KPNA-2 in the yeast two-hybrid system, and in particular with its C terminus. Unlike mutations in NLS-1 or NLS-2, which left the nuclear localization of Chk2 unaffected, mutations in NLS-3 caused a cytoplasmic relocalization, indicating that the NLS-3 motif acts indeed as NLS for Chk2 in vivo. Finally, co-transfection experiments with green fluorescent protein (GFP)-Chk2 and wild type or mutant KPNA-2 confirmed the role of KPNA-2 in nuclear import of Chk2.