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

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
?
Biochemical Pharmacology, Vol.60, pp.1143-1151,
2000
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C
N
NATURE INNUMOLOGY volume 9 number 3 march 2008
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Experimental Design

1. The ubiquitinating activity of A20
2. Characterization of A20 C-terminal ZnFs
   K48-linked ubiquitination its target, RIP its
   function, RIP proteasomal degradation
3. Characterization of A20 N-terminal OUT
   K63-linked DUB its target
4. 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

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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.
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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.
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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..
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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.
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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.
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Results
Fig2ab. 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.
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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.
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Results
Fig2de. 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.
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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.
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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
Fig3bc. 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.
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Results
Fig3def. 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.
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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.
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Results
Fig4ab. OTU deubiquitination is required for RIP
degradation
Fig4ab, both deletion (a) and mutation (b) of
the A20 OTU domain block destabilization of
co-transfected RIP in HEK293T cells.
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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.
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Results
Fig4de. 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.
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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.
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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

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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?

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