ABSTRACT
Inactivation of the TNFAIP3 gene, encoding the A20 protein, is associated with critical inflammatory diseases including multiple sclerosis, rheumatoid arthritis and Crohn's disease. However, the role of A20 in attenuating inflammatory signalling is unclear owing to paradoxical in vitro and in vivo findings. Here we utilize genetically engineered mice bearing mutations in the A20 ovarian tumour (OTU)-type deubiquitinase domain or in the zinc finger-4 (ZnF4) ubiquitin-binding motif to investigate these discrepancies. We find that phosphorylation of A20 promotes cleavage of Lys63-linked polyubiquitin chains by the OTU domain and enhances ZnF4-mediated substrate ubiquitination. Additionally, levels of linear ubiquitination dictate whether A20-deficient cells die in response to tumour necrosis factor. Mechanistically, linear ubiquitin chains preserve the architecture of the TNFR1 signalling complex by blocking A20-mediated disassembly of Lys63-linked polyubiquitin scaffolds. Collectively, our studies reveal molecular mechanisms whereby A20 deubiquitinase activity and ubiquitin binding, linear ubiquitination, and cellular kinases cooperate to regulate inflammation and cell death.
Subject(s)
Cysteine Endopeptidases/metabolism , Inflammation/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Ubiquitin/chemistry , Ubiquitin/metabolism , Animals , Cell Death , Cysteine Endopeptidases/chemistry , Cysteine Endopeptidases/genetics , Female , Inflammation/genetics , Inflammation/pathology , Intracellular Signaling Peptides and Proteins/chemistry , Intracellular Signaling Peptides and Proteins/genetics , Lysine/metabolism , Male , Mice , Mice, Inbred C57BL , Mutation , Phosphorylation , Polyubiquitin/chemistry , Polyubiquitin/metabolism , Protein Binding , Protein Kinases/metabolism , Signal Transduction , Tumor Necrosis Factor alpha-Induced Protein 3 , Tumor Necrosis Factor-alpha/metabolism , UbiquitinationABSTRACT
The engineering of biological components has been facilitated by de novo synthesis of gene-length DNA. Biological engineering at the level of pathways and genomes, however, requires a scalable and cost-effective assembly of DNA molecules that are longer than approximately 10 kb, and this remains a challenge. Here we present the development of pairwise selection assembly (PSA), a process that involves hierarchical construction of long-length DNA through the use of a standard set of components and operations. In PSA, activation tags at the termini of assembly sub-fragments are reused throughout the assembly process to activate vector-encoded selectable markers. Marker activation enables stringent selection for a correctly assembled product in vivo, often obviating the need for clonal isolation. Importantly, construction via PSA is sequence-independent, and does not require primary sequence modification (e.g. the addition or removal of restriction sites). The utility of PSA is demonstrated in the construction of a completely synthetic 91-kb chromosome arm from Saccharomyces cerevisiae.
