ABIN-1 is a ubiquitin sensor that restricts cell death and sustains embryonic development

Abstract

Proteins that directly regulate tumour necrosis factor receptor (TNFR) signalling have critical roles in regulating cellular activation and survival. ABIN-1 (A20 binding and inhibitor of NF-kappaB) is a novel protein that is thought to inhibit NF-kappaB signalling. Here we show that mice deficient for ABIN-1 die during embryogenesis with fetal liver apoptosis, anaemia and hypoplasia. ABIN-1 deficient cells are hypersensitive to tumour necrosis factor (TNF)-induced programmed cell death, and TNF deficiency rescues ABIN-1 deficient embryos. ABIN-1 inhibits caspase 8 recruitment to FADD (Fas-associated death domain-containing protein) in TNF-induced signalling complexes, preventing caspase 8 cleavage and programmed cell death. Moreover, ABIN-1 directly binds polyubiquitin chains and this ubiquitin sensing activity is required for ABIN-1's anti-apoptotic activity. These studies provide insights into how ubiquitination and ubiquitin sensing proteins regulate cellular and organismal survival.

Figure 1. ABIN-1 is required for embryonic development

(A) Gross appearance of ABIN-1+/+ and ABIN-1−/− embryos. (B) Hypoplasia of E18.5 ABIN-1−/− embryos. Weights of individual embryos are shown as circles; horizontal bars indicate mean weights for each genotype. ABIN-1−/− embryos weigh less than ABIN-1+/+ and ABIN-1+/− control embryos (p < 0.01 between ABIN-1+/+ and ABIN-1−/− embryos; and p < 0.01 between ABIN-1+/− and ABIN-1−/− embryos, n=35). (C) Anemia of E18.5 ABIN1−/− embryos. ABIN-1−/− embryos are markedly anemic (p < 0.02 between ABIN-1+/+ and ABIN-1−/− embryos; p < 0.01 between ABIN-1+/− and ABIN-1−/− embryos, n=18). (D) Hematoxilin and eosin (H&E) histology (upper panels) and cleaved caspase 3 immunohistochemistry (lower panels) of sequential sections from ABIN-1+/+ and ABIN-1−/− fetal livers. Apoptotic patches were found in 5 of 7 ABIN-1−/− fetal livers analyzed and in none of 10 ABIN-1+/− and ABIN+/+ fetal livers analyzed (p < 0.01).

Figure 2. ABIN-1 is required for protecting cells from TNF-induced PCD

(A) TNF-induced PCD of ABIN-1+/+ and ABIN-1−/− MEF (p < 0.01 between ABIN-1+/+ and ABIN-1−/− cells; means and standard deviations indicated, n=3). (B) TNF-induced caspase-3 and BID expression in ABIN-1+/+ (left) and ABIN-1−/− (right) MEFs. Cleaved, active forms of Bid (tBid) and caspase 3 indicated by arrows. Actin expression shown below as protein loading control. (C) TNF-induced PCD of ABIN-1−/− 3T3s expressing GFP and FLAG-ABIN-1 (black columns on right) or GFP alone (white columns on left), after treatment with the indicated agents. Means and standard deviations indicated, n=3. (D) TNF-induced caspase-8, caspase 3, and BID expression in ABIN-1−/− 3T3s expressing GFP and FLAG-ABIN-1 (left) or GFP alone (right). Cleaved, active forms of caspase 8, caspase 3 and Bid (tBid) indicated by arrows. Actin expression shown below as control. (E) TNF-induced expression of MAP kinase signaling proteins and A20 in ABIN-1−/− and control MEFs. (F) TNF-induced recruitment of RIP1 and FADD to Myc (ABIN-1). Expression of Myc-ABIN-1 in IPs, and expression of FADD and caspase 8 in whole cell lysates (“input”) shown below as controls. (G) TNF-induced recruitment of RIP1, FADD and A20 to caspase 8 in ABIN-1 deficient and control HT1080 cells. Expression of caspase 8 in IPs and expression of ABIN-1, FADD and actin in whole cell lysates (“input”) shown below as controls. All data are representative of 3–5 independent experiments.

Figure 3. ABIN-1 is a ubiquitin sensor that uses a NUB domain to bind to DISC signaling complexes and protect cells from TNF-induced PCD

(A) Sequence alignment of AHD2 (and neighboring sequences) of ABIN-1 with the NEMO ubiquitin binding (NUB) domain of NEMO/IKKγ. Thick bars mark locations of residues mutated in QQ477EE, F482S, and D485N ABIN-1 mutants. (B) Ubiquitin binding to GST-ABIN-1 in GST pull down assay. Ubiquitin bound to GST-ABIN-1 shown in left six lanes; input of ubiquitin chains is shown as a control in right three lanes. (C) GST-ABIN-1 proteins bound to K63-linked His-polyubiquitin chains in Nickel (Ni) agarose pull down assay. GST-ABIN-1 proteins bound to His-ubiquitin chains shown in upper panel; inputs of GST-ABIN-1 proteins shown in bottom panel as controls. (D) 6xHis-K63 linked ubiquitin chains bound to GST-C-terminal (wild type and mutant) ABIN-1 proteins. Input levels of GST-ABIN-1 proteins shown below as controls. (E) TNF-induced PCD of ABIN-1−/− 3T3s virally complemented with the indicated FLAG-ABIN-1 proteins or GFP alone. FLAG-ABIN-1 expression levels shown below as control. (F) Recruitment of RIP1 or FADD to wild type (WT) or mutant Myc-ABIN-1 proteins. Myc-ABIN-1 expression in anti-Myc immunoprecipitates and RIP1 and FADD expression in whole cell lysates (“input”) shown below as controls. (G) Recruitment of FADD to caspase 8 in presence of wild type, or QQ477EE mutant FLAG-ABIN-1 proteins, or no ABIN-1 proteins (“GFP”). Expression of caspase 8 in IPs and expression of FLAG-ABIN-1, caspase 8, FADD and actin in whole cell lysates (“input”) shown below as controls. (H) Recruitment of A20 to wild type or mutant (“QQ477EE”) FLAG-ABIN-1 proteins. Expression of FLAG-ABIN-1 in IPs and A20 in whole cell lysates (“input”) shown as controls. (I) Anti-apoptotic activity of ABIN-1 in A20−/− 3T3s expressing no ABIN-1 (“GFP”), WT ABIN-1 (“WT”), or mutant ABIN-1 (“QQ477EE”). Means and standard deviations indicated, n=3. Expression of FLAG-ABIN-1, A20, and actin proteins shown as controls at left (N.S. = non-specific band directly above FLAG-ABIN-1 band). All data are representative of 3–5 independent experiments.