CUL-2^LRR-1 and UBXN-3 drive replisome disassembly during DNA replication termination and mitosis

Replisome disassembly is the final step of DNA replication in eukaryotes, involving the ubiquitylation and CDC48-dependent dissolution of the CMG helicase (CDC45-MCM-GINS). Using Caenorhabditis elegans early embryos and Xenopus laevis egg extracts, we show that the E3 ligase CUL-2(LRR-1) associates with the replisome and drives ubiquitylation and disassembly of CMG, together with the CDC-48 cofactors UFD-1 and NPL-4. Removal of CMG from chromatin in frog egg extracts requires CUL2 neddylation, and our data identify chromatin recruitment of CUL2(LRR1) as a key regulated step during DNA replication termination. Interestingly, however, CMG persists on chromatin until prophase in worms that lack CUL-2(LRR-1), but is then removed by a mitotic pathway that requires the CDC-48 cofactor UBXN-3, orthologous to the human tumour suppressor FAF1. Partial inactivation of lrr-1 and ubxn-3 leads to synthetic lethality, suggesting future approaches by which a deeper understanding of CMG disassembly in metazoa could be exploited therapeutically

Bacteria-host relationship: Ubiquitin ligases as weapons of invasion

Eukaryotic cells utilize the ubiquitin (Ub) system for maintaining a balanced functioning of cellular pathways. Although the Ub system is exclusive to eukaryotes, prokaryotic bacteria have developed an armory of Ub ligase enzymes that are capable of employing the Ub systems of various hosts, ranging from plant to animal cells. These enzymes have been acquired through the evolution and can be classified into three main classes, RING (really interesting new gene), HECT (homologous to the E6-AP carboxyl terminus) and NEL (novel E3 ligases). In this review we describe the roles played by different classes of bacterial Ub ligases in infection and pathogenicity. We also provide an overview of the different mechanisms by which bacteria mimic specific components of the host Ub system and outline the gaps in our current understanding of their functions. Additionally, we discuss approaches and experimental tools for validating this class of enzymes as potential novel antibacterial therapy targets

Proapoptotic Bid Association with Mre11-Rad50-Nbs1 Complex is Indispensable for Checkpoint Activation after DNA Damage

AbstractThe proapoptotic protein Bid is phosphorylated by ATM after double strand breaks (DSBs) induction and induces S-phase arrest by a mechanism that remains to be elucidated. Here we show that in mammalian cells, Bid is associated with Mre11, a subunit of the Mre11-Rad50-Nbs1 (MRN) complex. We demonstrate that Bid activation is abrogated in Mre11 and Nbs1 deficient primary mouse fibroblasts and cells from patients with ataxia talangiectasia-like disorder. Bid depletion by RNA interference inhibited the S-phase checkpoint activation and G2/M arrest after genotoxic insult, but had no effect on MRN complex formation. Our results explain the mechanism of Bid phosphorylation by ATM in response to DNA damage and suggest that Bid functions as a link between the MRN complex and S-phase regulatory proteins.</jats:p

Autophagy regulates inflammatory programmed cell death via turnover of RHIM-domain proteins.

RIPK1, RIPK3, ZBP1 and TRIF, the four mammalian proteins harboring RIP homotypic interaction motif (RHIM) domains, are key components of inflammatory signaling and programmed cell death. RHIM-domain protein activation is mediated by their oligomerization; however, mechanisms that promote a return to homeostasis remain unknown. Here we show that autophagy is critical for the turnover of all RHIM-domain proteins. Macrophages lacking the autophagy gene Atg16l1accumulated highly insoluble forms of RIPK1, RIPK3, TRIF and ZBP1. Defective autophagy enhanced necroptosis by Tumor necrosis factor (TNF) and Toll-like receptor (TLR) ligands. TNF-mediated necroptosis was mediated by RIPK1 kinase activity, whereas TLR3- or TLR4-mediated death was dependent on TRIF and RIPK3. Unexpectedly, combined deletion of Atg16l1 and Zbp1 accelerated LPS-mediated necroptosis and sepsis in mice. Thus, ZBP1 drives necroptosis in the absence of the RIPK1-RHIM, but suppresses this process when multiple RHIM-domain containing proteins accumulate. These findings identify autophagy as a central regulator of innate inflammation governed by RHIM-domain proteins

Multiplexed proteomics of autophagy-deficient murine macrophages reveals enhanced antimicrobial immunity via the oxidative stress response.

Defective autophagy is strongly associated with chronic inflammation. Loss-of-function of the core autophagy gene Atg16l1 increases risk for Crohn's disease in part by enhancing innate immunity through myeloid cells such as macrophages. However, autophagy is also recognized as a mechanism for clearance of certain intracellular pathogens. These divergent observations prompted a re-evaluation of ATG16L1 in innate antimicrobial immunity. In this study, we found that loss of Atg16l1 in myeloid cells enhanced the killing of virulent Shigella flexneri (S.flexneri), a clinically relevant enteric bacterium that resides within the cytosol by escaping from membrane-bound compartments. Quantitative multiplexed proteomics of murine bone marrow-derived macrophages revealed that ATG16L1 deficiency significantly upregulated proteins involved in the glutathione-mediated antioxidant response to compensate for elevated oxidative stress, which simultaneously promoted S.flexneri killing. Consistent with this, myeloid-specific deletion of Atg16l1 in mice accelerated bacterial clearance in vitro and in vivo. Pharmacological induction of oxidative stress through suppression of cysteine import enhanced microbial clearance by macrophages. Conversely, antioxidant treatment of macrophages permitted S.flexneri proliferation. These findings demonstrate that control of oxidative stress by ATG16L1 and autophagy regulates antimicrobial immunity against intracellular pathogens