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De-ubiquitination and ubiquitin ligase domains of A20 downregulate NF- k B signalling

This study explores the de-ubiquitination and ubiquitination processes involved in the downregulation of NF-kB signalling by A20, a zinc finger protein. The study characterizes the different domains of A20 and their roles in ubiquitinating and de-ubiquitinating specific targets.

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De-ubiquitination and ubiquitin ligase domains of A20 downregulate NF- k B signalling

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  1. De-ubiquitination and ubiquitin ligase domains of A20 downregulate NF-kB signalling NATURE 430 694-699, 5 August 2004 Xiaoyuan Zhu 28 October 2008

  2. Introduction • De-ubiquitination & ubiquitination • NF-kB signaling • A20 (a zinc finger protein)

  3. De-ubiquitination & ubiquitinatation *DUBs: de-ubiquitinating enzymes

  4. NF-kB signaling Abbreviations: DD, death domain; FADD, FAS-associated DD protein; IkB, inhibitor of NF-kB; IKK, IkB-kinase; MAP3K, mitogen-activated protein kinase kinase kinase; NEMO, NF-kB essential modulator; NF-kB, nuclear factor-kB; RF, RING (really interesting new gene) finger; RNA pol II, RNA polymerase II; RIP, receptor-interacting protein; TNF: tumor necrosis factor; TNF-R1, TNF receptor 1; TRADD, TNF-receptor-associated DD protein; TRAF2, TNF-receptor-associated factor 2; Ub, ubiquitin; ZF, zinc finger TRENDS in Biochemical Sciences Vol.30 No.1 January 2005

  5. A20 ZnFs OTU-DUB ? Structure of A20 showing the location of the 7 zinc finger structures. The N-terminal A20 (ZF-) region is sufficient for binding to TRAF2 and TRAF6. The C-terminal A20 (ZF+) region, containing the 7 zinc finger structures, mediates A20 dimerization and the binding to several other proteins that might be involved in the inhibition of NF-kB activation and apoptosis by A20. Biochemical Pharmacology, Vol.60, pp.1143-1151, 2000

  6. C N NATURE INNUMOLOGY volume 9 number 3 march 2008

  7. Experimental Design The ubiquitinating activity of A20 Characterization of A20 C-terminal ZnFs: K48-linked ubiquitination; its target, RIP; its function, RIP proteasomal degradation Characterization of A20 N-terminal OUT: K63-linked DUB; its target Interplay between A20 C-terminal ZnFs and N-terminal OUT

  8. Methods • Cell culture, transfections and luciferase reporter assays • Immunoprecipitations and immunoblotting • In vivo ubiquitination and de-ubiquitination assays • In vitro ubiquitination assays • In vitro de-ubiquitination assays • RNA interference

  9. GenBank GI numbers A20 ZnF-4 A20 ZnF-5 Sequence conservation pattern A20 ZnF-7 Results S1. A multiple alignment of representatives of all versions of the A20 ZnF domains

  10. Results S2. The two distinct A20 domains and their connections to the Ub system Human: Trabid VCIP135 A20 Rabex-5/Rin-2 AWP1 C. elegans: F38B7.5 Yeast: Yf1044cp Abbreviations: An1-ZnF, the AN1-type Zinc finger; Ub, ubiquitin; UBA/CUE, small, a-helical ubiquitin binding domain; RAB-GEF, Vps9-type exchange factor for RAB GTP-ases; LF, little finger domain; UBCH, Ub hydrolase; C2H2-U, a specialized version of the C2H2 ZnF implicated in Ub signaling. A20 possibly participates in ubiquitin signaling pathways.

  11. Results Fig.1a. A20 or interacting proteins catalyses polyubiquitination with specific E2s Fig.1a. In vitro ubiquitination assays with FLAG-eluted A20 from HEK293T cells and a panel of ubiquitin conjugating (E2) enzymes. Ubiquitinated proteins are detected by anti-biotin western blot (WB). T6: TRAF6, a positive control ubiquitin ligase (E3) for hUBC13/UEV2-mediated polyubiquitination.

  12. Results Fig.1bc. A20 catalyses polyubiquitination with the C-terminal ZnF domain Fig.1b, A2 FLAG-eluted from HEK293T cells or purified from E. coli has intrinsic ubiquitin ligase activity in vitro when all reaction components are supplied. C, A20 ZnFs catalyse in vitro polyubiquitination. A20 proteins were purified from E. coli..

  13. Results S3, S4. ZnFs 3 and/or 4 are required for polyubiquitination catalysis S3, S4. In vitro ubiquitination assays with A20 truncation mutants. A20 proteins were FLAG-eluted from HEK293T cells. Deletional analysis maps the A20 Ub ligase domain to ZnFs 3 and 4.

  14. Results S5, Fig1d. ZnF4, but not ZnF3, is important for polyubiquitination catalysis S5, In vitro ubiquitination assays with A20 point mutants. A20 proteins were FLAG-eluted from HEK293T cells. Point mutations of conserved cysteines in A20 ZnF4, but not ZnF3, reduce in vitro ubiquitination catalyzed by A20. b. A20 proteins were purified from E. coli. All markers on the left hand side are in kDa.

  15. Results Fig2a&b. A20 catalyses K48-linked polyubiquitination Fig2a, Schematic diagram of ubiquitin mutants. b, A20 purified from E. coli autoubiquitinates with K48-only, but not K63-only, polyubiquitin chains in vitro.

  16. Results S6, Fig2c. RIP (receptor interacting protein) is an A20 substrate C? S6, TNFR1-recruited RIP is ubiquitinated and targeted for proteasomal degradation. HelaS3 cells were treated with TNF-a (TNF Rx) for the indicated time, with or without MG-132 pre-treatment. Endogenous TNFR1 immunoprecipitates were then blotted with an anti-RIP antibody to reveal TNFR1-associated RIP. Fig2c, A20 purified from E. coli directly ubiquitinates RIP purified from insect cell lysates in vitro. * MG132, the proteasome inhibitor.

  17. Results Fig2d&e. A20-mediated RIP degradation are dependent on ZnF4 Fig2d, Co-transfection of A20 promotes RIP degradation in HEK293T cells. A20 ZnF4MT, but not ZnF3MT, attenuates RIP degradation. E, Endogenous RIP binding to transfected FLAG-A20 variants in HEK293T cells.

  18. Results Fig2f. A20 downregulates TNF-a-induced NF-kB signalling in A20-/- MEFs via ZnF4 Fig2f, Complementation of A20-/- MEFs with wild-type, but not ZnF4MT, A20 effectively downregulates NF-kB signalling. Average values are of four independent transfection. *MEFs, murine embryonic fibroblasts.

  19. Results Fig3a. The N-terminal OTU domain of A20 is a K48-de-ubiquitinating enzyme (DUB) Fig3a, A20 purified from E. coli depolymerizes free polyubiquitin chains in vitro. * OTU, ovarian tumour.

  20. Results Fig3b&c. RIP is a substrate for the A20 N-terminal OUT domain Fig3b, Co-transfection of wild-type A20 de-ubiquitinates RIP in HEK293T cells. c, Transfected wild-type and OTU mutant FLAG-A20 bind endogenous RIP equally well in HEK293T cells.

  21. Results Fig3d&e&f. A20 directly removes TRAF2-mediated K63-linked RIP ubiquitination Fig3d, A20 purified from E. coli de-ubiquitinates FLAG-RIP purified from HEK293T cells in vitro. NEM, N-ethylmaleimide, a DUB inhibitor. e, Transfection of TRAF2 specifically promotes K63-linked ubiquitination of endogenous RIP in HEK293T cells. f, Ubiquitination of transfected RIP is reduced by attenuation of endogenous TRAF2 or hUBC13 expression with small interfering RNA oligonucleotides (siRNA) in HEK293T cells.

  22. Results Fig3g. NF-kB downregulation requires OTU-mediated de-ubiquitination in A20-/- MEFs Fig3g, A20-/- MEFs was complemented with wild-type A20 or the A20 OTU catalytic cysteine mutant (C103A). Then NF-kB activity and A20 expression were measured. Anti-actin staining was a loading control.

  23. Results Fig4a&b. OTU deubiquitination is required for RIP degradation Fig4a&b, both deletion (a) and mutation (b) of the A20 OTU domain block destabilization of co-transfected RIP in HEK293T cells.

  24. Results Fig4c. OTU deubiquitination is a prerequisite for ZnF4 polyubiquitination Fig4c, Functional interplay of the A20 OTU and ZnF4 domains in co-transfected HEK293T cells. De-ubiquitination of K63-linked chains on RIP by the A20 OTU domain is a prerequisite for RIP polyubiquitination with K48-linked chains by A20 ZnF4.

  25. Results Fig4d&e. Both OTU and ZnF4 are essential in regulating TNFR1-associated RIP degradation and TNF-a-induced NF-kB activity Fig4d, Endogenous RIP recruited to TNFR1 is hyperubiquitinated and stabilized in A20-/- MEFS. e, Both the A20 OTU domain and the A20 ZnF4 domains regulate the stability of TNFR1-associated endogenous RIP in complemented A20-/- MEFs.

  26. S7. Proposed mechanism of A20-mediated NF-kB downregulation. Step 1: proximal signaling proteins are recruited to the activated TNFR1; a/b/g: IkK complex. Step 2: TRAF2-mediated K63-linked polyubiquitination (black diamonds) stabilizes association of proximal signaling components, thereby promoting IkK complex activation. Step 3: A20 removes K63-linked Ub chains from RIP, which is a prerequisite for Step 4. Step : A20 polyubiquitinates RIP with K48-linked chains (grey ovals). Step 5: K48-ubiquitinated RIP is targeted for proteasomal degradation, thereby terminating NF-kB signaling.

  27. Conclusion A20 downregulates NF-kB signaling through the cooperative activity of its two ubiquitin-editing domains. The C-terminal ZnF4 domain of A20 functions as a ubiquitin ligase by polyubiquitinating RIP with K48-linked ubiquitin chains, thereby targeting RIP for proteasomal degradation. The N-terminal OTU domain of A20 functions as a deubiquitinating enzyme (DUB) by removing K63-linked ubiquitin chains from RIP, which is a prerequisite for ZnF4-mediated K48-linked ubiquitination. Both OTU and ZnF4 cooperate to downregulate NF-kB signaling

  28. PROs and CONs • PROs • The organization is simple and clear • The experimental design and results are convincing • The background and discussion are sufficient • CONs • The experimental design and results are needed to be simplified • Why A20-/- murine embryonic fibroblasts, but not A20-/- mice embryonic fibroblasts?

  29. A20 most possibly participates in ubiquitin signaling pathways. In vitro ubiquitination assays to check ubiquitin ligase activity of A20 K63/K48; Identify A20 substrate

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