Human papillomavirus type 16 E6 and E7 oncoproteins interact with the nuclear p53-binding protein 1 in an in vitro reconstructed 3D epithelium: new insights for the virus-induced DNA damage response.

BACKGROUND: Despite vaccination and screening measures, anogenital cancer, mainly promoted by HPV16 oncoproteins, still represents the fourth tumor and the second cause of death among women. Cell replication fidelity is the result of the host DNA damage response (DDR). Unlike many DNA viruses that promote their life cycle through the DDR inactivation, HR-HPVs encourage cells proliferation despite the DDR turned on. Why and how it occurs has been only partially elucidated. During HPV16 infection, E6 links and degrades p53 via the binding to the E6AP LXXLL sequence; unfortunately, E6 direct role in the DDR response has not clearly identified yet. Similarly, E7 increases DDR by competing with E2F1-pRb interaction, thus leading to the inactivation of pRb, and promotion, E2F1 mediated, of DDR genes translation, by binding to the pRb-like proteins CBP/p300 and p107, that also harbour LXXLL sequence, and via the interaction and activation of several DDR proteins. METHODS: To gain information regarding E6 and E7 contribution in DDR activation, we produced an in vitro 3D HPV16-E6E7 infected epithelium, already consolidated study model for HPVs, and validated it by assessing H&E staining and BrdU, HPV16 DNA, E6E7 proteins and γH2A.X/53BP1 double-strand break (DSBs) sensors expression; then we made an immuno-colocalization of E6 and E7 with cyclin E2 and B1. Since 53BP1, like E6 and E7, also binds p53 and pRb, we supposed their possible direct binding. To explore this hypothesis, we performed a double immunofluorescence of E6 and E7 with 53BP1, a sequence analysis of 53BP1 within its BRCT2 domain and then an in situ PLA within CaSki, E6E7HPV16 NHEKs and the 3D model. RESULTS: The in vitro epithelium resembled the histology and the events typical of in vivo infected tissues. E6E7HPV16 were both expressed in basal and differentiated strata and induced H2A.X phosphorylation and 53BP1 increment into nuclear foci. After highlighting E6 and E7 co-expression with 53BP1 and a LKVLL sequence within the 53BP1 BRCT2 domain, we demonstrated the bindings via the PLA technique. CONCLUSIONS: Our results reinforce E6 and E7 role in cellular function control providing potentially new insights into the activity of this tumor virus.

RNF168 promotes noncanonical K27 ubiquitination to signal DNA damage.

Ubiquitination regulates numerous cellular processes by generating a versatile communication system based on eight structurally and functionally different chains linked through distinct residues. Except for K48 and K63, the biological relevance of different linkages is largely unclear. Here, we show that RNF168 ubiquitin ligase promotes noncanonical K27-linked ubiquitination both in vivo and in vitro. We demonstrate that residue K27 of ubiquitin (UbK27) is required for RNF168-dependent chromatin ubiquitination, by targeting histones H2A/H2A.X, and that it is the major ubiquitin-based modification marking chromatin upon DNA damage. Indeed, UbK27 is strictly required for the proper activation of the DNA damage response (DDR) and is directly recognized by crucial DDR mediators, namely 53BP1, Rap80, RNF168, and RNF169. Mutation of UbK27 has dramatic consequences on DDR activation, preventing the recruitment of 53BP1 and BRCA1 to DDR foci. Similarly to the DDR, atypical ubiquitin chains could play unanticipated roles in other crucial ubiquitin-mediated biological processes.

A novel ubiquitin mark at the N-terminal tail of histone H2As targeted by RNF168 ubiquitin ligase.

Ubiquitination of histones plays a critical role in the regulation of several processes within the nucleus, including maintenance of genome stability and transcriptional regulation. The only known ubiquitination site on histones is represented by a conserved Lys residue located at the C terminus of the protein. Here, we describe a novel ubiquitin mark at the N-terminal tail of histone H2As consisting of two Lys residues at positions 13 and 15 (K13/K15). This "bidentate" site is a target of the DNA damage response (DDR) ubiquitin ligases RNF8 and RNF168. Histone mutants lacking the K13/K15 site impair RNF168- and DNA damage-dependent ubiquitination. Conversely, inactivation of the canonical C-terminal site prevents the constitutive monoubiquitination of histone H2As but does not abolish the ubiquitination induced by RNF168. A ubiquitination-defective mutant is obtained by inactivating both the N- and the C-terminal sites, suggesting that these are unique, non-redundant acceptors of ubiquitination on histone H2As. This unprecedented result implies that RNF168 generates a qualitatively different Ub mark on chromatin.

UMI, a novel RNF168 ubiquitin binding domain involved in the DNA damage signaling pathway.

Ubiquitination regulates important cellular processes, including the DNA damage response (DDR) and DNA repair. The complexity of the ubiquitin-mediated signals is decoded by ubiquitin receptors, which contain protein modules named ubiquitin binding domains (UBDs). We previously identified a new ubiquitin ligase, RNF168, involved in DDR and endowed with two UBDs named MIU (motif interacting with ubiquitin). Here we have provided the identification of a novel UBD, the UMI (UIM- and MIU-related UBD), present in RNF168, and characterized the interaction surface with ubiquitin, centered on two Leu residues. We have demonstrated that integrity of the UMI, in addition to the MIUs, is necessary for the proper localization of RNF168 and for ubiquitination of nuclear proteins, including histone H2A. Finally, we have shown that simultaneous inactivation of UMI and MIUs prevents the recruitment to DDR foci of the crucial downstream mediator 53BP1.

RNF168, a new RING finger, MIU-containing protein that modifies chromatin by ubiquitination of histones H2A and H2AX.

BACKGROUND: Modulation of chromatin structure has emerged as a critical molecular device to control gene expression. Histones undergo different post-translational modifications that increase chromatin accessibility to a number of regulatory factors. Among them, histone ubiquitination appears relevant in nuclear processes that govern gene silencing, either by inhibiting or activating transcription, and maintain genome stability, acting as scaffold to properly organize the DNA damage response. Thus, it is of paramount importance the identification and the characterization of new ubiquitin ligases that address histones. RESULTS: We identified and characterized RNF168, a new chromatin-associated RING finger protein. We demonstrated that RNF168 is endowed with ubiquitin ligase activity both in vitro and in vivo, which targets histones H2A and H2AX, but not H2B, forming K63 polyubiquitin chains. We previously described the presence within RNF168 sequence of two MIU domains, responsible for the binding to ubiquitinated proteins. Here we showed that inactivation of the MIUs impairs ubiquitin binding ability in vitro and reduces chromatin association of RNF168 in vivo. Moreover, upon formation of DNA double strand breaks induced by chemical and physical agents, RNF168 is recruited to the DNA damage foci, where it co-localizes with gammaH2AX and 53BP1. The localization of RNF168 at the site of damage highly increases the local concentration of ubiquitinated proteins and determines the prolonged ubiquitination signal. CONCLUSION: The RING finger protein RNF168 is a new ubiquitin ligase that functions as chromatin modifier, through histone ubiquitination. We hypothesize a dual function for RNF168. In normal condition RNF168 modifies chromatin structure by modulating ubiquitination of histone H2A. Upon DNA lesions, RNF168 is recruited to DNA damage response foci where it contributes to increase the amount of ubiquitinated proteins, thereby facilitating the downstream signalling cascade.

Preliminary data suggestive of a novel translational approach to mesothelioma treatment: imatinib mesylate with gemcitabine or pemetrexed.

BACKGROUND: Malignant mesothelioma is a cancer which is refractory to current treatments. Imatinib mesylate is a selective inhibitor of tyrosine kinases such as bcr-abl, c-Kit, c-Fms and platelet derived growth factor receptor beta (PDGFRbeta). PDGFRbeta is often overexpressed in mesothelioma cells and is a therapeutic target for imatinib in some solid tumours. A study was undertaken to assess whether imatinib alone or combined with chemotherapeutic agents may be effective for treating mesothelioma. METHODS: Cultures from mesothelioma MMP, REN and ISTMES2 cell lines were treated with imatinib alone or in combination with a chemotherapeutic agent. RESULTS: Imatinib induced cytotoxicity and apoptosis selectively on PDGFRbeta positive mesothelioma cells via blockade of receptor phosphorylation and interference with the Akt pathway. Of the chemotherapeutic agents tested in combination with imatinib, a synergistic effect was obtained with gemcitabine and pemetrexed. CONCLUSIONS: This study provides a rationale for a novel translational approach to the treatment of mesothelioma which relies on enhancement of tumour chemosensitivity by inhibition of Akt.