The study of death receptor (DR) signaling has led to the discovery of new signaling paradigms, including the first example of direct receptor-mediated activation of a protease (caspase-8) that ...functions as a second messenger to initiate a 'death cascade' of downstream protease activation. More recently, this receptor system has underscored the importance of ubiquitin modification in NF-kappaB activation. Both degradative lysine 48-linked polyubiquitin and scaffolding lysine 63-linked polyubiquitin have an essential role in signal propagation. Remarkably, a negative feedback process, termed ubiquitin editing, regulates signaling that emanates from certain DRs. Ubiquitin editing is mediated by a complex interplay between the ubiquitination and deubiquitination machinery, resulting in the replacement of signal enhancing lysine 63-linked polyubiquitin with signal extinguishing lysine 48-linked polyubiquitin. The ubiquitination machinery and its regulation in the context of DR signaling are discussed herein.
Arabidopsis thaliana De-etiolated-1 (AtDET1) is a highly conserved protein, with orthologs in vertebrate and invertebrate organisms. AtDET1 negatively regulates photomorphogenesis, but its ...biochemical mechanism and function in other species are unknown. We report that human DET1 (hDET1) promotes ubiquitination and degradation of the proto-oncogenic transcription factor c-Jun by assembling a multisubunit ubiquitin ligase containing DNA Damage Binding Protein-1 (DDB1), cullin 4A (CUL4A), Regulator of Cullins-1 (ROC1), and constitutively photomorphogenic-1. Ablation of any subunit by RNA interference stabilized c-Jun and increased c-Jun-activated transcription. These findings characterize a c-Jun ubiquitin ligase and define a specific function for hDET1 in mammalian cells.
Posttranslational modification of proteins with polyubiquitin occurs in diverse signaling pathways and is tightly regulated to ensure cellular homeostasis. Studies employing ubiquitin mutants suggest ...that the fate of polyubiquitinated proteins is determined by which lysine within ubiquitin is linked to the C terminus of an adjacent ubiquitin. We have developed linkage-specific antibodies that recognize polyubiquitin chains joined through lysine 63 (K63) or 48 (K48). A cocrystal structure of an anti-K63 linkage Fab bound to K63-linked diubiquitin provides insight into the molecular basis for specificity. We use these antibodies to demonstrate that RIP1, which is essential for tumor necrosis factor-induced NF-κB activation, and IRAK1, which participates in signaling by interleukin-1β and Toll-like receptors, both undergo polyubiquitin editing in stimulated cells. Both kinase adaptors initially acquire K63-linked polyubiquitin, while at later times K48-linked polyubiquitin targets them for proteasomal degradation. Polyubiquitin editing may therefore be a general mechanism for attenuating innate immune signaling.
NF-κB transcription factors mediate the effects of pro-inflammatory cytokines such as tumour necrosis factor-α and interleukin-1β. Failure to downregulate NF-κB transcriptional activity results in ...chronic inflammation and cell death, as observed in A20-deficient mice. A20 is a potent inhibitor of NF-κB signalling, but its mechanism of action is unknown. Here we show that A20 downregulates NF-κB signalling through the cooperative activity of its two ubiquitin-editing domains. The amino-terminal domain of A20, which is a de-ubiquitinating (DUB) enzyme of the OTU (ovarian tumour) family, removes lysine-63 (K63)-linked ubiquitin chains from receptor interacting protein (RIP), an essential mediator of the proximal TNF receptor 1 (TNFR1) signalling complex. The carboxy-terminal domain of A20, composed of seven C2/C2 zinc fingers, then functions as a ubiquitin ligase by polyubiquitinating RIP with K48-linked ubiquitin chains, thereby targeting RIP for proteasomal degradation. Here we define a novel ubiquitin ligase domain and identify two sequential mechanisms by which A20 downregulates NF-κB signalling. We also provide an example of a protein containing separate ubiquitin ligase and DUB domains, both of which participate in mediating a distinct regulatory effect.
Apoptosis inhibition rather than enhanced cellular proliferation occurs in prostate cancer (CaP), the most commonly diagnosed malignancy in American men. Therefore, it is important to characterize ...residual apoptotic pathways in CaP cells. When intracellular Ca2+ stores are released and plasma membrane “store-operated” Ca2+ entry channels subsequently open, cytosolic Ca2+ increases and is thought to induce apoptosis. However, cells incapable of releasing Ca2+ stores are resistant to apoptotic stimuli, indicating that Ca2+ store release is also important. We investigated whether release of intracellular Ca2+ stores is sufficient to induce apoptosis of the CaP cell line LNCaP. We developed a method to release stored Ca2+ without elevating cytosolic Ca2+; this stimulus induced LNCaP cell apoptosis. We compared the apoptotic pathways activated by intracellular Ca2+ store release with the dual insults of store release and cytosolic Ca2+ elevation. Earlier processing of caspases-3 and -7 occurred when intracellular store release was the sole Ca2+ perturbation. Apoptosis was attenuated in both conditions in stable transfected cells expressing antiapoptotic proteins BclxL and catalytically inactive caspase-9, and in both scenarios inactive caspase-9 became complexed with caspase-7. Thus, intracellular Ca2+ store release initiates an apoptotic pathway similar to that elicited by the dual stimuli of cytosolic Ca2+ elevation and intracellular store release.
The experimental study and manipulation of programmed cell death has been greatly assisted by the identification of genetic and pharmacological tools that can either induce or block cell lethality. ...This review discusses new insights into the molecular sensing of perturbations induced by such tools, as well as the possible consequences of this detection in determining cell survival.
The post-translational modification of proteins with ubiquitin represents a complex signalling system that co-ordinates essential cellular functions, including proteolysis, DNA repair, receptor ...signalling and cell communication. DUBs (deubiquitinases), the enzymes that disassemble ubiquitin chains and remove ubiquitin from proteins, are central to this system. Reflecting the complexity and versatility of ubiquitin signalling, DUB activity is controlled in multiple ways. Although several lines of evidence indicate that aberrant DUB function may promote human disease, the underlying molecular mechanisms are often unclear. Notwithstanding, considerable interest in DUBs as potential drug targets has emerged over the past years. The future success of DUB-based therapy development will require connecting the basic science of DUB function and enzymology with drug discovery. In the present review, we discuss new insights into DUB activity regulation and their links to disease, focusing on the role of DUBs as regulators of cell identity and differentiation, and discuss their potential as emerging drug targets.
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