Localized protein biotinylation at DNA damage sites identifies ZPET, a repressor of homologous recombination.

Numerous DNA repair and signaling proteins function at DNA damage sites to protect the genome. Here, we show that fusion of the promiscuous biotin ligase BirAR118G with RAD18 leads to localized protein biotinylation at DNA damage sites, allowing identification of ZPET (zinc finger protein proximal to RAD eighteen)/ZNF280C as a potential DNA damage response (DDR) protein. ZPET binds ssDNA and localizes to DNA double-strand breaks (DSBs) and stalled replication forks. In vitro, ZPET inhibits MRE11 binding to ssDNA. In cells, ZPET delays MRE11 binding to chromatin after DSB formation and slows DNA end resection through binding ssDNA. ZPET hinders resection independently of 53BP1 and HELB. Cells lacking ZPET displayed enhanced homologous recombination (HR), accelerated replication forks under stress, and increased resistance to DSBs and PARP inhibition. These results not only reveal ZPET as an HR repressor but also suggest that localized protein biotinylation at DNA damage sites is a useful strategy to identify DDR proteins.

Increased virus dissemination leads to enhanced lung injury but not inflammation during influenza-associated secondary bacterial infection.

Secondary bacterial infections increase influenza-related morbidity and mortality, particularly if acquired after 5-7 d from the viral onset. Synergistic host responses and direct pathogen-pathogen interactions are thought to lead to a state of hyperinflammation, but the kinetics of the lung pathology have not yet been detailed, and identifying the contribution of different mechanisms to disease is difficult because these may change over time. To address this gap, we examined host-pathogen and lung pathology dynamics following a secondary bacterial infection initiated at different time points after influenza within a murine model. We then used a mathematical approach to quantify the increased virus dissemination in the lung, coinfection time-dependent bacterial kinetics, and virus-mediated and postbacterial depletion of alveolar macrophages. The data showed that viral loads increase regardless of coinfection timing, which our mathematical model predicted and histomorphometry data confirmed was due to a robust increase in the number of infected cells. Bacterial loads were dependent on the time of coinfection and corresponded to the level of IAV-induced alveolar macrophage depletion. Our mathematical model suggested that the additional depletion of these cells following the bacterial invasion was mediated primarily by the virus. Contrary to current belief, inflammation was not enhanced and did not correlate with neutrophilia. The enhanced disease severity was correlated to inflammation, but this was due to a nonlinearity in this correlation. This study highlights the importance of dissecting nonlinearities during complex infections and demonstrated the increased dissemination of virus within the lung during bacterial coinfection and simultaneous modulation of immune responses during influenza-associated bacterial pneumonia.

CYLD deubiquitinates RIP1 in the TNFα-induced necrosome to facilitate kinase activation and programmed necrosis.

BACKGROUND: Necroptosis/programmed necrosis is initiated by a macro-molecular protein complex termed the necrosome. Receptor interacting protein kinase 1 (RIPK1/RIP1) and RIP3 are key components of the necrosome. TNFα is a prototypic inducer of necrosome activation, and it is widely believed that deubiquitination of RIP1 at the TNFR-1 signaling complex precedes transition of RIP1 into the cytosol where it forms the RIP1-RIP3 necrosome. Cylindromatosis (CYLD) is believed to promote programmed necrosis by facilitating RIP1 deubiquitination at this membrane receptor complex. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate that RIP1 is indeed the primary target of CYLD in TNFα-induced programmed necrosis. We observed that CYLD does not regulate RIP1 ubiquitination at the TNF receptor. TNF and zVAD-induced programmed necrosis was highly attenuated in CYLD(-/-) cells. However, in the presence of cycloheximide or SMAC mimetics, programmed necrosis was only moderately reduced in CYLD(-/-) cells. Under the latter conditions, RIP1-RIP3 necrosome formation is only delayed, but not abolished in CYLD(-/-) cells. We further demonstrate that RIP1 within the NP-40 insoluble necrosome is ubiquitinated and that CYLD regulates RIP1 ubiquitination in this compartment. Hence, RIP1 ubiquitination in this late-forming complex is greatly increased in CYLD(-/-) cells. Increased RIP1 ubiquitination impairs RIP1 and RIP3 phosphorylation, a signature of kinase activation. CONCLUSIONS/SIGNIFICANCE: Our results show that CYLD regulates RIP1 ubiquitination in the TNFα-induced necrosome, but not in the TNFR-1 signaling complex. In cells sensitized to programmed necrosis with SMAC mimetics, CYLD is not essential for necrosome assembly. Since SMAC mimetics induces the loss of the E3 ligases cIAP1 and cIAP2, reduced RIP1 ubiquitination could lead to reduced requirement for CYLD to remove ubiquitin chains from RIP1 in the TNFR-1 complex. As increased RIP1 ubiquitination in the necrosome correlates with impaired RIP1 and RIP3 phosphorylation and function, these results suggest that CYLD controls RIP1 kinase activity during necrosome assembly.