1. The K48-K63 Branched Ubiquitin Chain Regulates NF-κB Signaling
Fumiaki Ohtake, Yasushi Saeki, Satoshi Ishido, Jun Kanno, Keiji Tanaka 
Molecular Cell 
Volume 64, Issue 2, Pages (October 2016)
DOI: /j.molcel
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2. Molecular Cell 2016 64, 251-266DOI: (10.1016/j.molcel.2016.09.014)
Copyright © 2016 Elsevier Inc. Terms and Conditions

3. Figure 1 Identification of K48-K63 Branched Ubiquitin Chains
(A) Relative amounts of eight types of polyubiquitin linkages in whole-cell lysates from untreated U2OS_shUb cells. Data are derived from lane 1 of Figure 2D.
(B) Schematic representation of heterogenous ubiquitin chains. Branched, mixed, and multiple chains are indistinguishable in mass-spectrometric analysis.
(C) The ubiquitin R54A mutation enables identification and quantification of K48-K63 branched linkages, unbranched K48 linkages (not branched at K63), and unbranched K63 linkages (not branched at K48).
(D) Point mutations in R54 do not affect abundance of K48 or K63 chains in cells. 293F cells were transfected with the indicated FLAG-Ub derivatives and immunoprecipitated using anti-FLAG antibody. IP, immunoprecipitation.
(E) Identification of K48-K63 branched ubiquitin chains. 293F cells were transfected with FLAG-Ub(R54A), and anti-FLAG immunoprecipitants were subjected to mass-spectrometric analysis. The MS/MS spectrum corresponding to the signature peptide derived from K48-K63 branched linkages (aa 43–72, GlyGly modified at K48 and K63) was obtained.
(F) Identification of endogenous K48-K63 branched ubiquitin chains. 293F cell lysates were immunoprecipitated with anti-Ub (FK2), and the precipitants were digested with fewer amounts of trypsin. Using PRM, MS/MS fragment ions derived from the signature peptide mis-cleaved at Arg54 (aa 43–72, GlyGly modified at K48 and K63) were observed with the same retention time.
See also Figures S1 and S7 and Table S1.
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4. Figure 2 Quantification of K48-K63 Branched Ubiquitin Linkage in Cells
(A) Ubiquitin replacement strategy for wild-type and R54A ubiquitin. U2OS_shUb_rUb(WT) and U2OS_shUb_rUb(R54A) cells were incubated with doxycycline (Dox, 1 ng/ml) for 5 days, and whole-cell lysates were analyzed by western blotting. In the upper panel, rUb(WT) and rUb(R54A) are expressed at similar levels. In the lower panel, the replaced rUb is expressed at a similar level to endogenous ubiquitin in intact cells.
(B) Strategy for quantifying K48-unbranched K63 linkages derived from Ub(R54A). (i) Scheme for enzymatic conversion of N-terminal Gln in a peptide into pyroGlu. (ii) Immunoprecipitated Ub(R54A) was subjected to in-gel trypsin digestion. For the QPCT(+) sample, peptides were incubated with QPCT. Total peak areas of selected MS/MS fragment ions derived from Gln-containing or pyroGlu-containing signature peptide from an unbranched K63 linkage (aa 55–72, GlyGly modified at K63) were compared.
(C) Co-elution of light (sample-derived) and heavy (AQUA) peptides for absolute quantification. Ub was immunoprecipitated from Dox-treated U2OS_shUb_rUb(R54A) cells and subjected to a PRM analysis.
(D) The K48-K63 branched ubiquitin linkage is abundant in cells. Dox-treated U2OS_shUb_rUb(R54A) cells were treated/untreated with 20 μM MG132 for 1 hr, and whole-cell lysates were subjected to a PRM analysis. Signature peptides specific to endogenous Ub (i) or replaced Ub(R54A) (ii), or common to both (iii), were quantified. (iv) Total K63 linkages containing branched, unbranched, and endogenous (un-replaced) species were present at comparable levels in Dox-treated and untreated cells. Error bars indicate means ± SEM (n = 3).
See also Figure S2.
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5. Figure 3 IL1β-Dependent Assembly of the K48-K63 Branched Chain
(A) IL1β-dependent assembly of polyubiquitin chains on endogenous TRAF6 complex. Dox-treated U2OS_shUb_rUb(R54A) cells were treated with IL1β for 20 min and immunoprecipitated using anti-TRAF6 antibody. IP, immunoprecipitation; IB, immunoblot; WCL whole-cell lysate.
(B) IL1β-dependent assembly of the K48-K63 branched chain on TRAF6. (i) Immunopurified TRAF6 in (A) was subjected to PRM analysis. The data were normalized against the amount of total ubiquitin chains in each experiment. Error bars indicate means ± SEM (n = 4). (ii) Percentage of branched and unbranched K63 linkages were calculated for each experiment. Error bars indicate means ± SEM (n = 4).
(C) (i) Quality control of specificity of DUBs used in the assay. (ii) TRAF6 immunoprecipitated from U2OS cells treated with IL1β for 20 min were subjected to ubiquitin restriction assay with AMSH, OTUB1, and USP2. Cleavage of ubiquitin chains were analyzed by immunoblotting. (iii) The density of smears in (ii) were quantified.
(D) Catalytic activity of TRAF6 is required for K48-K63 branch assembly. FLAG-TRAF6, either wild-type or bearing the C70A mutation, was co-expressed with HA-Ub(R54A) in 293F cells, and immunopurified TRAF6 was subjected to PRM quantification. Error bars indicate means ± SD (n = 3 for WT, and n = 2 for C70A).
(E) Confirmation of catalytic deficiency of TRAF6(C70A). Recombinant TRAF6 (WT or C70A) (1 μg) was incubated with E1 (50 ng), UBC13 (50 ng), UEV1A (50 ng), and Ub(R54A) (2 μg) for 3.5 hr at 30°C in an in vitro ubiquitylation assay and immunoblotted using anti-Ub antibody (Dako).
(F) 293F cells were transfected with FLAG-TRAF6 and HA-Ub(R54A), treated with MG132 for 2 hr and subjected to immunoprecipitation using anti-FLAG antibody. Error bars indicate means ± SEM (n = 3).
See also Figure S3.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 4
HUWE1 Assembles K48-K63 Branched Ubiquitin Chains in Cooperation with TRAF6
(A) Identification of HUWE1 as a TRAF6-interacting protein. FLAG-TRAF6 (WT or C70A)-associated proteins were immunoprecipitated from 293F cells and identified by LC-MS. The difference in spectrum counts (PSM) identified from TRAF6(WT) and the PSM identified from TRAF6(C70A) is plotted versus the PSM from TRAF6(WT). The red line indicates 2-fold enrichment of PSM in TRAF6(WT). A replicate experiment is shown in Figure S4A.
(B) Endogenous HUWE1 associates with catalytically active TRAF6. FLAG-TRAF6-interacting proteins from 293F cells were analyzed using the indicated antibodies. IP, immunoprecipitation.
(C) Endogenous HUWE1 interacts with IRAK1 via TRAF6. Cells were transfected with FLAG-IRAK1 and HA-TRAF6 as indicated and were immunoprecipitated with anti-FLAG antibody.
(D) HUWE1 mainly assembles K48 chains in vitro. Recombinant HUWE1HECT (0.3 μg) was incubated with E1 (50 ng), UbcH7 (50 ng), and a single-lysine Ub mutant (5 μg) for 30 min at 30°C in an in vitro ubiquitylation assay. The products were immunoblotted using anti-Ub antibody (Dako).
(E) HUWE1 assembles K48-K63 branched ubiquitin chains in cooperation with TRAF6 in vitro. Ub(R54A) (2 μg) was incubated with TRAF6 (0.4 μg), UBC13 (100 ng), UEV1A (100 ng), and E1 (50 ng) for 4 hr; mixed with HUWE1 HECT (50–100 ng) and UbcH7 (100 ng); and incubated for an additional 30 min. The samples were analyzed by PRM. Error bars indicate means ± SD (n = 2).
(F) HUWE1 interacts with K63-chain-modified TRAF6 through the UBA-UIM domain. Recombinant GST-TRAF6 (WT or C70A) bound on glutathione sepharose was incubated with/without E1, Ubc13-Uev1a, Ub, and ATP. After washing, purified HUWE1 (WT or ΔUBA/UIM) was added, and a GST pull-down assay was performed.
(G) The HUWE1-UBA/UIM domain interacts with K63-linked Ub2–4 but not with monoubiquitin. K63 chains were synthesized using Ub(WT) or R54A using Ubc13-Uev1a, and a GST pull-down assay was performed using the indicated recombinant ubiquitin-binding domains. IB, immunoblot.
(H) Recombinant GST (glutathione S-transferase)-TRAF6 was incubated with/without E1, Ubc13-Uev1a, and Ub(R54A) in an in vitro ubiquitylation assay for 1 hr. After washing, purified HUWE1 (WT or ΔUBA/UIM) and UbcH7 were used for the second reaction for 1 hr. Branched linkage formation was quantified. Error bars indicate means ± SEM (n = 3).
(I) Branched chain formation as in (H), but TRAF6-Ubc13/Uev1a or HUWE1-UbcH7 was sequentially added in the indicated orders. After the first and second reactions, GST-TRAF6 bound to beads was washed to remove enzymes. Error bars indicate means ± SEM (n = 3).
See also Figure S4 and Table S2.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 5 HUWE1 Positively Regulates NF-κB Signaling
(A) HUWE1 regulates the K48-K63 branched chain as well as the K63 and M1 chains. U2OS_shUb_rUb(R54A) cells transfected with siHUWE1 or a scrambled siRNA were subjected to ubiquitin replacement by Dox. The cells were then treated with IL1β for 20 min and immunoprecipitated using anti-TRAF6 antibody. After PRM analyses, the data were normalized against the amount of total ubiquitin chains in each experiment and presented as the change relative to siScramble. Error bars indicate means ± SD (n = 2). HUWE1 knockdown efficiency is shown in the right panel.
(B) HUWE1 is a positive regulator of NF-κB-mediated gene induction. Untreated U2OS_shUb cells were transfected with two independent HUWE1 siRNAs, TRAF6 siRNA, or a scrambled siRNA for 3 days and then treated with IL1β for 1 hr; RNAs were isolated for qRT-PCR. Data were normalized against β-actin. Error bars indicate means ± SEM (n = 3).
(C) HUWE1 is a positive regulator of NF-κB activity. 293F cells were transfected with the indicated siRNAs for 1 day. The cells were then transfected with NF-κB reporter plasmids and the indicated expression vectors and incubated for an additional 24–26 hr before cell lysis for luciferase assay. Error bars indicate means ± SEM (n = 3).
(D) HUWE1 positively regulates IL1β-induced IκBα phosphorylation. Untreated U2OS_shUb cells were transfected with the indicated siRNAs for 72 hr and then treated with IL1β for the indicated periods. Whole-cell lysates were analyzed by western blotting.
(E) Interaction of TRAF6 with TAB2/TAK1 complex and modification with K63-polyubiquitin are impaired by HUWE1 knockdown. 293F cells transfected with the indicated siRNAs for 24 hr were transfected with FLAG-TRAF6 and HA-Ub(R54A) for an additional 2 days. Cell lysates were immunoprecipitated with anti-FLAG antibody and then analyzed by immunoblotting or PRM quantification of branched linkages. Right panel shows means ± SEM (n = 3). IP, immunoprecipitation; WCL, whole-cell lysate.
See also Figure S5.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 6
The K48-K63 Branch Counteracts CYLD-Mediated K63 Deubiquitylation
(A and B) GST-TRAF6 (0.5 μg) bound to glutathione beads was incubated first with Ubc13-Uev1a and subsequently with or without HUWE1HECT (0.1 μg or 1.0 μg (++)) in an in vitro ubiquitylation assay. After washing the beads, the resultant K63 chains or K48-K63 branched chains conjugated to 0.1 μg TRAF6 were subjected to deubiquitylation assay with (A) 0.05 μg CYLD or (B) 0.5 μg A20 phosphorylated by IKKβ for the indicated periods. The K63 chains were blotted using anti-K63ub antibody.
(C) Distal K48 branching inhibits cleavage of the K63 linkage by CYLD. K63-diUb (0.25 μg) or K48/63/48-tetraUb (0.5 μg) was incubated with recombinant CYLD (10 ng) at 37°C for the indicated periods. Ubiquitin and ubiquitin chains were stained with SYPRO Ruby. Relative intensities of the bands are plotted in the lower panel.
(D) The K48 branch does not affect recognition of K63-diubiquitin by TAB2. K48-K63 branched triubiquitin, as well as K48- or K63-linked diubiquitin, was synthesized using donor and acceptor ubiquitin and E2 enzymes UBC13-UEV1A and/or UBE2K, as detailed in Figure S6D. Pull-down assay was performed using GST-tagged TAB2-NZF as bait. Bound ubiquitin was blotted using anti-Ub antibody. TAB2-NZF was stained with SYPRO Ruby (lower panel).
(E) The effect of HUWE1 knockdown on association of TRAF6 with TAB2/TAK1 is abolished by CYLD double knockdown. 293F cells transfected with the indicated siRNAs were transfected with FLAG-TRAF6 (lanes 2–5) and HA-Ub (lanes 1–5). The cells were subjected to anti-FLAG immunoprecipitation (IP). WCL, whole-cell lysate.
(F and G) The effect of HUWE1 knockdown on NF-κB activity (F) and target gene induction (G) is abolished by CYLD double knockdown. (F) 293F cells were transfected with the indicated siRNAs and plasmids, as in Figure 5C, and subjected to luciferase assay. Error bars indicate means ± SEM (n = 6). (G) Untreated U2OS_shUb cells were transfected with the indicated siRNAs, and qRT-PCR was performed as in Figure 5B. Error bars indicate means ± SEM (n = 3).
See also Figure S6.
Molecular Cell  , DOI: ( /j.molcel )
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9. Figure 7
The K48-K63 Branched Ubiquitin Chain Is an Abundant Modification and a Positive Regulator of NF-κB Signal
Mass-spectrometry-based quantification revealed that the K48-K63 branched ubiquitin chain is an abundant modification in cells. During IL1β signaling, the E3 ubiquitin ligase HUWE1 assembles K48-K63 branched ubiquitin chains in cooperation with TRAF6, which enhances NF-κB activation by inhibiting CYLD-mediated K63 chain disassembly while preserving recognition by TAB2 for the downstream cascade. We propose that ubiquitin chain branching constitutes a new component of the ubiquitin code, adding additional diversity to ubiquitin biology.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2016 Elsevier Inc. Terms and Conditions