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Figure 1 - Human HOIP is ubiquitinated at K784 and regulates NF-κB.

A. A scheme of procedures for the UbiCRest-based assays employed to analyze HOIP-modification

with ubiquitin chains. Total cell extracts from HEK293T cells transiently expressing Myc-HOIP and

Myc-OTULIN C129A, a catalytic inactive mutant, subjected to GST-linear-TUBE pulldown followed

by UbiCRest using recombinant deubiquitinases (vOTU, OTULIN and USP21).

B. UbiCRest assays to evaluate ubiquitin chain types on HOIP examined by immunoblotting.

Immunoblotting of samples using antibodies as indicated. Ponceau S staining used for monitoring GSTliner-TUBE input. Representative data shown from three independent experiments.

C. Mass spectrometry spectra corresponding to a peptide containing HOIP-K784 with double Gly

(114+K).

D. Domains of human HOIP and identified ubiquitination sites at K454, K458, K735 and K784.

E. Multiple sequence alignment of different HOIP orthologues illustrating the position K735 and K784

according to the ClustalX colour scheme. Sequences were retrieved from the NCBI protein database

with the following accessions: Homo sapiens (NP_060469.4), Canis lupus (XP_005623312.1), Mus

musculus

(NP_919327.2),

(NP_001090429.1),

Monodelphis

Alligator

domestica

mississippiensis

(XP_007479924.1),

(XP_006259801.1),

Xenopus

Takifugu

laevis

rubripes

(XP_003968217.2) and Drosophila melanogaster (NP_723214.2).

F. Co-immunoprecipitation analysis of the HOIP K784R mutant with SHARPIN and HOIL-1L using

total cell extracts of HEK293T cells transiently expressing Myc-HOIP wildtype (WT), or Myc-HOIPK784R with HOIL-1L-HA and Flag-SHARPIN. Anti-Vinculin antibody used to monitor protein

loading. Representative data shown from three independent experiments.

G. Luciferase-based NF-κB gene reporter assays using Myc -HOIP wildtype (WT), a catalytic inactive

mutant C885A, ubiquitination site mutants of K454R, K458R, K735R, K784R co-transfected with

HOIL-1L-HA and Flag-SHARPIN. Luciferase signal was normalized to an internal Renilla control

signal.

Data information: In (G), data are presented as mean ± SD. **p≤0.01, ****p≤0.0001 (ANOVA). n=4.

27 Figure 2 - The human HOIP K784R mutant as a part of the LUBAC generates linear ubiquitin

chains and ubiquitinates its substrate NEMO in vitro and in cells.

A. In vitro ubiquitination assays for the indicated times using the recombinant proteins of ubiquitin

(Ub), E1, E2 (UbcH7), HOIP (WT, K784R or C885A mutant), HOIL-1L, SHARPIN and NEMO.

Immunoblotting of Linear ubiquitin chains, NEMO, HOIP, HOIL-1L and SHARPIN detected by using

antibodies as indicated. Representative data from three independent experiments.

B. Immunoblotting to detect ubiquitination of NEMO in HEK293T cells transiently expressing FlagNEMO, GFP-SHARPIN, and HOIL-1L-HA with Myc-HOIP wildtype (WT), Myc-HOIP K784R or

Myc-HOIP C885A. Total cell lysates in denaturing conditions subjected to SDS-PAGE followed up by

immunoblotting using antibodies as indicated. Anti-Vinculin antibody to monitor loading.

Representative data from three independent experiments.

Figure 3 –No obvious developmental defect in HoipK778R/K778R knockin mice is observed while TNFresponses are suppressed in HoipK778R/K778R cells.

A. Numbers of weaned mice of the indicated genotypes from Hoip+/K778R crosses.

B. A gross appearance image of Hoip+/+ and HoipK778R/K778R male mice at 6-week old.

C,D. Immunoblotting to detect TNF-induced degradation and phosphorylation of IB- or

phosphorylation of IKK in immortalized Hoip+/+ and HoipK778R/K778R MEFs treated with human TNF

(20ng/ml) for the indicated times. Immunoblots of anti-Vinculin antibody and anti--Tubulin antibody

shown for monitoring loading amount. Representative data from three independent experiments.

E. Induction of TNF-dependent NF-κB target genes, ICAM, VCAM and IB- in Hoip+/+ or

HoipK778R/K778R immortalized MEFs determined by qRT-PCR. RNA extraction and cDNA synthesis

from MEFs treated with hTNF (20ng/ml) for the indicated time subjected to examine transcripts of

ICAM, VCAM and IB-. Normalization to β-actin. Representative data from three independent

experiments, n=3.

F. TNF-dependent induction of cleavage of PARP and Caspase 3 in primary Hoip+/+ or HoipK778R/K778R

MEFs determined by immunoblotting. Total cell extracts of MEFs treated with hTNF (100ng/ml) and

CHX (1µg/ml) for the indicated times subjected to SDS-PAGE followed by immunoblotting using

antibodies as indicated. Anti--Tubulin antibody used to monitor loading amount. Representative data

of two independent experiments.

G. TNF-dependent induction of Caspase 3 activation in immortalized Hoip+/+ or HoipK778R/K778R MEFs

measured by using DEVD-AFC. MEFs treated with hTNF (100ng/ml) and CHX (1µg/ml) for 4 hours

subjected to the Caspase 3 activity assays.

28 H. TNF-induced Caspase 8 activity in Hoip+/+ or HoipK778R/K778R immortalized MEFs treated with hTNF

(100ng/ml) with or without Cycloheximide (CHX) (1µg/ml) or z-VAD (20µM). Representative data

from three independent experiments, n=4.

I. TNF-receptor complex II formation in Hoip+/+ or HoipK778R/K778R immortalized MEFs. Total cell

extracts of MEFs treated with hTNF (100ng/ml), CHX (1µg/ml) and zVAD (25µM) for the indicated

time immunoprecipitated using an anti-FADD antibody. Recruitment of RIPK1, HOIP and SHARPIN

monitored by immunoblotting. The anti-Vinculin antibody blot shown for monitoring loading amount.

The black arrows indicating HOIP and SHARPIN in the IP samples and unmodified RIPK in the input

samples, respectively. Representative data of two independent experiments.

Data information: In (E,G and H), data are presented as mean ± SD. **p≤0.01, ***p≤0.001,

****p≤0.0001 (ANOVA, n=4 in G).

CHX (Cycloheximide), hTNF (human Tumor Necrosis Factor), z-VAD (Z-Val-Ala-Asp fluoromethyl

ketone)

Figure 4 - SHARPIN-deficiency leads HoipK778R/K778R mice to embryonic lethality, which is rescued

by TNFR1 knockout.

A. Numbers of weaned mice of the indicated genotypes from crosses of Hoip+/K778R; Sharpin+/cpdm mice.

B. Number of the embryos of the indicated genotype at E12.5 and E13.5 from crosses of HoipK778R/K778R;

Sharpin+/cpdm mice.

C. Gross appearance images of HoipK778R/K778R; Sharpin+/+, HoipK778R/K778R; Sharpin+/cpdm and

HoipK778R/K778R; Sharpincpdm/cpdm embryos at E13.5. Representative pictures from 7 embryos each. Scale

bars: 1 mm.

D. Gross appearance images of Hoip+/K778R;Sharpincpdm/cpdm;Tnfr1+/-and HoipK778R/+; Sharpincpdm/cpdm ;

Tnfr1-/- female mice at 6 weeks of age, and a female HoipK778R/K778R; Sharpincpdm/cpdm ; Tnfr1-/- mouse at

8 weeks of age.

E. H&E staining, Keratin 14 (KRT14) and Cleaved Caspase 3 immunostaining of dorsal skin sections

from mice of the indicated genotypes at 4 or 8 weeks of age. Scale bars: 50 µm.

Figure 5 – SHARPIN-deficiency in Hoip+/K778R heterozygous mice leads to early onset of skin

inflammation accompanied with apoptosis induction.

29 A. Gross appearance of mice of the indicated genotypes (male mice at 4-week old). Scale bars: 10 mm.

B. Immunostaining (H&E, Keratin 14 (KRT14), Ly6G, F4/80 and Cleaved Caspase 3) of mouse dorsal

skin sections of the indicated genotypes. Scale bars: 50 µm.

C-E. Measurements (Total epidermis skin thickness, Keratin layer thickness and Squamous cell layer

thickness) of dorsal skin sections obtained from male mice of the indicated genotypes. Each dot on the

scatter dot plot represents one focus point of the measurement. (N=20, 20, 30, 40, 40 for Hoip+/+;

Sharpin+/+, HoipK778R/K778R; Sharpin+/+ , Hoip+/K778R; Sharpin+/cpdm,

Hoip+/+; Sharpincpdm/cpdm,

Hoip+/K778R; Sharpincpdm/cpdm, respectively.)

F. Immunoblotting to examine TNF-induced degradation and phosphorylation of IκB-α in immortalized

MEFs of the indicated genotypes using total cell extracts of MEFs treated with hTNF (20ng/ml) for the

indicated times. Representative of three independent experiments.

G. Immunoblotting of TNF-induced cleavage of PARP and caspase 3 in immortalized MEFs of the

indicated genotypes using total cell extracts of MEFs treated with hTNF (100ng/ml) with or without

CHX (1µg/ml) for indicated times. Representative of three independent experiments.

H,I. TNF-dependent induction of Caspase 3 activation in immortalized MEFs of the indicated genotype

measured by DEVD-AFC. MEFs treated with hTNF (100ng/ml) alone, or with CHX (1µg/ml) for 4

hours (H) or 2 hours (I) subjected to the Caspase 3 activity assays. A representative data of two

independent experiments, n=4.

J. TNF-induced Caspase 8 activity in immortalized MEFs of the indicated genotype. Luminoldependent activity of Caspase 8 in immortalized MEFs treated with hTNF (100ng/ml) with or without

CHX (1µg/ml) . A representative data of three independent experiments, n=4.

Data information: In (C-E and H-J), data are presented as mean ± SD. **p≤0.01 , ***p≤0.001,

****p≤0.0001, ANOVA.

hTNF (human Tumor Necrosis Factor), CHX (Cycloheximide),

Figure 6 – A proposed model of HOIP-site specific ubiquitination in the regulation of the TNF

signaling cascades.

A working model describing a role of site-specific ubiquitination of HOIP in the regulation of the TNFinduced NF-B and apoptosis pathways based on this study. Schematics of simplified TNF signaling

in wild type cells (WT, left panel) and HOIP-K784R cells (right panel) are shown. Upon TNF-binding

to TNF receptor 1, complex I, which includes RIPK1 and LUBAC components (HOIP, HOIL-1L and

SHARPIN) is formed. In this signaling cascade, LUBAC linearly ubiquitinates NEMO and RIPK1. Site

30 specific ubiquitination of HOIP at K784 plays a role in inducing TNF-dependent NF-B target genes

and in inhibition of apoptosis by inhibiting RIPK1 recruitment into TNF receptor complex II. Linkage

types of ubiquitin chains conjugated on HOIP-K784 remains open.

31 ...