www.sciencemag.org/cgi/content/full/science.aau1208/DC1

Supplementary Materials for

N-terminal degradation activates the NLRP1B inflammasome

Ashley J. Chui*, Marian C. Okondo*, Sahana D. Ra*, Kuo Gai, Andrew R. Griswold,

Darren C. Johnson, Daniel P. Ball, Cornelius Y. Taabazuing, Elizabeth L. Orth,

Brooke A. Vittimberga, Daniel A. Bachovchin†

*These authors contributed equally to this work.

†Corresponding author. Email: bachovcd@mskcc.org

Published 14 March 2019 on Science First Release

DOI: 10.1126/science.aau1208

This PDF file includes:

Materials and Methods

Figs. S1 to S14

Tables S1 to S3

Captions for Data S1 and S2

References

Other Supplementary Material for this manuscript includes the following:

(available at www.sciencemag.org/cgi/content/full/science.aau1208/DC1)

Data S1 and S2

2

Materials and Methods

Cloning. sgRNAs were designed using the Broad Institute’s web portal (29)

(http://www.broadinstitute.org/rnai/public/analysis-tools/sgrna-design) and cloned into the

lentiGuide-puro vector (Addgene #52963) as described previously (30). The sgRNA sequences

are listed in Table S3. cDNA encoding the full-length mouse Nlrp1b gene was cloned from RAW

264.7 macrophages and moved into the pDEST27 vector (Addgene) or a modified pLEX_307

vector (Addgene) with an N-terminal V5-GFP tag and C-terminal FLAG tag using Gateway

technology (Thermo Fisher Scientific). The L45M point mutation was generated using the

QuickChange site-directed mutagenesis kit (Agilent). The indicated mNlrp1b fragments were

subcloned into the pDEST27 vector or into modified pLEX_307 vectors with a C-terminal FLAG

or Myc tags. Where indicated, the Nlrp1b fragments were fused in frame to ubiquitin and/or GFP

before subcloning into a modified pLEX_307 vector with a C-terminal FLAG tag. cDNA

encoding for human CARD8 was purchased from Origene and was subcloned into a modified

pLEX_307 vector with a C-terminal FLAG tag. cDNA coding for full length human GSDMD

(Dharmacon) was cloned into the pLEX_307 vector (Addgene) with a C-terminal V5 tag. cDNA

encoding for full length human CASP1 (Origene) and mouse Casp1 (Origene) were cloned with

stop codons into a modified version of the pLEX_307 vector (Addgene) with a hygromycin

resistance marker. A plasmid for expressing mouse Ubr2 (pcDNA3.1-His vector backbone) was

provided by Yong Tae Kwon (Seoul National University). The following siRNA sequences were

used: UBR2: 5ʹ-CAACUACAGUAGAUCGAGA-3ʹ; UBR4 5ʹ-GCAGAUCCAUCAACUACGA-

3ʹ.

3

Reagents and antibodies. Val-boroPro (31) and compound 8j (32) were synthesized according to

previously published protocols. For cell culture experiments, Val-boroPro was resuspended in

DMSO containing 0.1% TFA to prevent compound cyclization. Anthrax lethal toxin was

purchased from List Biological Laboratories; bortezomib from LC laboratories; bestatin methyl

ester, L-phenylalaninamide hydrochloride (L-Phe-NH2), amastatin, and cycloheximide from

Sigma-Aldrich; CHR-2797 from Tocris Bioscience; batamistat from R&D Systems; bestatin from

VWR; and actinonin from Enzo Life Sciences. Antibodies used include: mouse anti-mouse

caspase-1 at 1 μg/ml (AG20B-0042, Adipogen), rabbit anti-GAPDH at 1:1000 (clone 14C10, Cell

Signaling Technology), mouse anti-V5 at 1 μg/mL (V5-10, Sigma-Aldrich), mouse anti-GST at

1:1000 (26H1, Cell Signaling Technology), mouse anti-Myc at 1:1000 (9B11, Cell Signaling

Technology), mouse anti-FLAG M2 at 2 μg/mL (F1804, Sigma-Aldrich), rabbit anti-HA at 1

μg/mL (ab9110, Abcam), rabbit anti-human/mouse UBR2 at 1 μg/mL (ab217069, Abcam), rabbit

anti-mouse UBA6 at 1:500 (#13386, Cell Signaling Technology), rabbit anti-human/mouse UBR4

at 1 μg/mL (ab86738, Abcam), rabbit anti-mouse DNAJA2 at 1 μg/mL (ab157216, Abcam),

rabbit anti-mouse ACTR5 at 1:1000 (21505-1-AP, Proteintech), rabbit anti-mouse INO80b at

1:500 (ab175117, Abcam), rabbit anti-mouse ACTR8 at 2 μg/mL (ab177335, Abcam), rabbit anti-

human PARP at 1:500 (#9542, Cell Signaling Technology), rabbit anti-human GSDMD at 1:500

(NBP2-33422, Novus Biologicals), rabbit anti-human CARD8 N-terminal at 1:500 (ab194585,

Abcam), rabbit anti-human CARD8 C-terminal at 2 μg/mL (ab24186, Abcam), rabbit anti-human

Actin at 1:1000 (13E5, Cell Signaling Technology), and mouse anti-mouse NLRP1B (2A12) at

1:250 (this monoclonal antibody was a gift from the Vance laboratory at UC Berkeley).

4

Cell culture. HEK 293T and RAW 264.7 cells were purchased from ATCC and grown in

Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS). THP-1 cells

were purchased from ATCC and grown in RPMI-1640 medium with 10% FBS. All cells were

grown at 37 °C in a 5% CO2 incubator. Cell lines were regularly tested for mycoplasma using the

MycoAlertä Mycoplasma Detection Kit (Lonza).

LDH cytotoxicity assays. RAW 264.7 cells were seeded at 0.5 ´ 106 cells/well in six-well plates

in standard growth medium 24 h prior to treatment. Cells were treated with the indicated agents

for the indicated times. Supernatants were then harvested and analyzed for LDH activity using an

LDH cytotoxicity assay kit (Pierce). For experiments with proteasome inhibitors, bestatin methyl

ester, and L-Phe-NH2, cells were treated with these agents 30 min prior to the addition of Val-

boroPro and/or LT, and then incubated for the indicated amount of time. For LDH release

experiments in HEK 293T cells, we used cells stably expressing mCASP1, mCASP1 and

mGSDMD, or human CASP1 and hGSDMD developed previously (8, 25). The indicated HEK

293T cells were then seeded at 0.5 × 106 cells/well in six-well plates in standard growth medium,

and 24 h later transiently transfected with indicated constructs encoding Nlrp1b (or fragments) or

CARD8 using Fugene HD. After 24 h, supernatants were then harvested and analyzed for LDH

activity, or cells were treated with the indicated agents for an additional 6 h before LDH release

was assessed.

CellTiter-Glo cell viability assay. DPP8/9–/– and CASP1–/– THP-1 cells were generated

previously (7). THP-1 and RAW 264.7 cells were plated (2,000 cells/well) in white, 384-well clear

bottom plates (Corning) using an EL406 Microplate Washer/Dispenser (BioTek) in a 25 μL final

5

volume of media. Compounds were added to THP-1 cells using a pintool (CyBio). THP-1 cells

were incubated for 48 h at 37 °C, and RAW 264.7 cells were incubated for the indicated times at

37 °C. Assay plates were then removed from the incubator and allowed to equilibrate to room

temperature on the bench top before addition of 10 µL of CellTiter-Glo reagent (Promega)

according to the manufacturer’s instructions. Assay plates were shaken on an orbital shaker for 2

min and incubated at room temperature on the bench top for 10 min. Luminescence was then read

using a Cytation 5 Cell Imaging Multi-Mode Reader (BioTek).

Knockout cell lines. RAW 264.7 and HEK 293T cells stably expressing Cas9 were generated

previously (7). Constructs encoding sgRNAs were packaged into lentivirus in HEK 293T cells

using the Fugene HD transfection reagent (Promega) and 2 µg of the vector, 2 µg PAX2 (Addgene,

#35002), and 1 µg pMD2.G (Addgene, #12259), and incubated for 48 h. RAW 264.7 and HEK

293T were spinfected with virus-containing supernatant from the transfection for 2 h at 1000 ´ g

at 30 °C (HEK 293T cells were treated with 8 µg/mL polybrene during spinfection). After 2 d,

cells were selected for stable expression of sgRNAs using puromycin (5 µg/mL for RAW 264.7

cells, 1 µg/mL for HEK 293T cells). After 10 d, single cells were isolated by serial dilution and

expanded to obtain complete knockouts. RAW 264.7 cells treated with sgRNAs targeting Actr5

were incubated with VbP (10 µM) for 1 week to obtain ACTR5-deficient cells.

Cytokine stimulation in mice. For cytokine induction in C57BL/6J mice (The Jackson

Laboratory), 9-week-old male mice were injected intraperitoneally with 100 µL of vehicle (1 N

HCl in PBS, pH = 7.4), 100 µL of Val-boroPro (20 µg/ 100 µL), 100 µL of bestatin (50 mg/kg),

100 µL of bestatin methyl ester (50 mg/kg), or the indicated combinations. Val-boroPro was stored

6

at 10× final concentration in 0.01N HCl and diluted into PBS immediately before dosing. Bestatin

and bestatin methyl ester was stored at 10× final concentration in DMSO and diluted into PBS

immediately before dosing. Serum was collected 6 h after dosing via retro-orbital collection and

G-CSF levels were measured by Quantikine ELISA (R&D Systems). This animal protocol was

reviewed and approved by the Memorial Sloan Kettering Cancer Center Institutional Animal Care

and Use Committee (IACUC). Sample sizes were based on the statistical analysis of previous

experiments with vehicle-treated mice versus Val-boroPro-treated mice (7, 33). No animals were

excluded. The experiments were not randomized and the investigators were not blinded.

Immunoblotting experiments. RAW 264.7 macrophages were seeded at 0.5 × 106 cells/well in

six-well plates or at 5 × 106 cells/dish in 10 cm plates, and 24 h later were treated with agents as

described. Bestatin methyl ester and bortezomib, if used, were added 30 min prior to LT or VbP

treatment. For experiments using HEK 293T cells, cells were seeded at 0.5 × 106 cells/well in six-

well plates, and 24 h later transiently transfected with the indicated constructs using Fugene HD.

After 24 h, lysates were then harvested, or cells treated with agents as indicated before lysates were

harvested. For siRNA experiments, RNAiMax Transfection reagent was used to transfect

HEK293T cells with 30 nM of GAPDH-cy3 (Thermofisher Scientific AM4611), UBR2, and/or

UBR4 siRNAs using manufacturer’s instructions. Cells were incubated at 37 °C for 24 h before

transfecting with 0.05 µg of the NLRP1B-GFP constructs using Fugene HD and incubating for an

additional 24 h at 37 °C before harvesting cells for analysis. Lysates were normalized to 1 mg/mL

using the DC Protein Assay kit (Bio-Rad), separated by SDS–PAGE, immunoblotted, and

visualized using the Odyssey Imaging System (Li-Cor).

7

Immunoprecipitation experiments. For immunoprecipitation experiments, HEK 293T cells were

seeded at 0.5 × 106 cells/well in six-well plates and transiently transfected with the indicated

plasmids using Fugene HD. After 18 h, cells were then treated with the indicated agents for an

additional 6 h and harvested. Cells were lysed in lysis buffer (0.5% NP-40 in 1× TBS) and

normalized to 1 mg/mL using the DC Protein Assay kit (Bio-Rad). Immunoprecipitations were

carried out by incubating 1 mg of total cell lysate with 40 µL of anti-FLAG-M2 agarose resin

(Sigma-Aldrich) in a 1 mL total volume overnight at 4 °C. Beads were then washed 3× in PBS

(pH 7.4), and bound proteins were eluted in 3× FLAG peptide (150 ng/µL). The samples were

separated by SDS–PAGE, immunoblotted, and visualized using the Odyssey Imaging System (Li-

Cor).

FACS Analysis. Cells treated as indicated were harvested and washed twice in 1× PBS. Pellets

were suspended in a 1× PBS buffer containing 2.5% FBS, 150 µg/mL DNAse I (150 µg/mL), and

5 mM MgCl2. Cells were stained with DAPI (Thermofisher Scientific) and were sorted using a BD

Fortessa instrument (BD Bioscience). Data were analyzed using FACSDiva (BD Bioscience) and

FlowJo software. GFP-positive and GFP-negative cells were gated out of the total population of

cells, single cells, and live cells.

In vitro Lethal Factor cleavage assay. HEK 293T cells were seeded at 0.5 × 106 cells/well in

six-well plates and transiently transfected with 2 µg of the indicated Nlrp1b plasmids using

FuGENE HD. After 24 h, lysates were harvested and normalized to 1 mg/mL using the DC Protein

Assay kit (Bio-Rad). These lysates were then incubated with LF (10 ng/mL) for up to 2 h at room

8

temperature. Aliquots were removed and quenched by boiling with SDS–PAGE sample buffer at

the indicated times. Samples were evaluated by immunoblotting.

Genome wide CRISPR- Cas9 Knockout Screens. Lentiviral Production: The mouse GeCKO

2-vector system sgRNA pool B library (Addgene, # 1000000053) was amplified and verified by

the RNAi core at MSKCC following established protocols (34). For lentiviral production, HEK

293T cells were seeded at 15 × 106 cells in 4 × 15 cm dishes in DMEM with 10% FBS. Twenty-

four hours later, cells were transfected with 15 µg Pax2 and 10 µg pMD2.G (Addgene # 35002

and 12259, respectively) using 75 µL of Fugene HD reagent in 1.5 mL OPTI-MEM. Media was

replaced 6-8 h after transfection with DMEM containing 30% FBS. Two days later, lentivirus was

harvested, aliquoted, and frozen at –80 °C. Viral titer was measured following established

protocols (12). Briefly, RAW 264.7 cells stably expressing Cas9 (7) were seeded at 3 × 106

cells/well in six-well plates. A range of virus-containing supernatant (0-1,000 µL) was added and

the multiplicity of infection (MOI) was determined by calculating the number of transduced cells

3 d after selection with puromycin. VbP and LT selection screens: 180 × 106 RAW 264.7 cells

stably expressing Cas 9 were seeded at 3 × 106 cells/well in six-well plates. The pooled library was

transduced at an MOI of 0.3 with a coverage of >500 cells per sgRNA. Plates were spinfected at

1,500 × g for 90 min and incubated for 72 h. Cells were then selected with puromycin (5 µg/ml)

for 14 d. The transduced cells were seeded at 5 × 107 cells for each replicate of control, VbP (2

µM) and LT (1 µg/mL) treatments (n = 3 per treatment). The untreated cells were harvested 24 h

after seeding. VbP-treated cells were treated with drug for 1 week before being washed 3 × with

PBS (10 mL) and propagated for an additional 5 d before harvesting. For LT selection, cells were

9

treated with 1 µg/mL each of protective antigen (PA) and lethal factor (LF) for 3 h. Cells were

washed twice with PBS and allowed to propagate for 4 d after replacing media. Cells were then

selected similarly with LT for a second time. Cells were harvested at seeding concentration and

cell pellets frozen. Illumina library preparation: Frozen cell pellets were thawed and genomic

DNA was harvested using the DNeasy blood and tissue kit (Qiagen # 69506). A two-step PCR

protocol (12) with Phusion high fidelity DNA polymerase (New England BioLabs) was performed

following the manufactured protocol (NEB M0530). We used established primer sequences for

amplifying the Lentiguide puro sgRNA for the first PCR (12) and standard Illumina primers and

adaptor sequences for the second PCR (Addgene). The amplicons were extracted from 2% agarose

gel using the QIAquick Gel Extraction Kit (Qiagen # 28076). The samples were quantified, pooled

and sequenced on a Hiseq2500 by the Integrated Genomics Operation at MSKCC. RIGER

analysis: A pseudocount of one was added to all read counts for each sgRNA in each treatment

group. Each sgRNA was normalized to its relative representation in each sample. Fold-

enrichment was determined by dividing the average sgRNA representation in the treated samples

by the average sgRNA representation in the control samples. RIGER analysis (35) was performed

using GENE-E software (https://software.broadinstitute.org/GENE-E/) using the Second Best

Rank algorithm that takes into account the combined sum of the first and second best ranks for

sgRNAs for a given gene, as described on the GENE-E website.

10

Fig. S1. Schematic of the genome-wide CRISPR/Cas9 screens. Different colored cells indicate infection with different sgRNAs.

Pooled sgRNA library

Lentivirusproduction

Library packaged in lentivirus

Transduced RAW 264.7 cells

VbPSelection

LTSelection

sequenced ascontrol

VbP-resistantRAW 264.7 cells

LT-resistantRAW 264.7 cells

Viralinfection

sequenced forVbP resistance

sgRNAs

sequenced forLT resistance

sgRNAs

shared resistancesgRNAs

VbP resistance onlysgRNAs

LT resistance onlysgRNAs

11

Fig. S2. Dnaja2–/– RAW 264.7 cells are resistant to VbP and LT. (A) Confirmation of Dnaja2 knockout in RAW 264.7 cells by immunoblotting. Cells were treated with DMSO, VbP, or compound 8j for 24 h prior to immunoblotting. (B, C) Control or Dnaja2 KO RAW 264.7 macrophages were treated with VbP (2 µM, 24 h) or 8j (20 µM, 24 h) (B) or LT (1 µg/mL, 6 h) (C) before cytotoxicity was determined by LDH release. Data are means ± SEM of three biological replicates. ***p < 0.001; *p < 0.05; NS, not significant by two-sided Student’s t-test. Data are representative of three independent experiments.

+- -- -+ +- -

GFP_

sg1

Val-boroPro (2 μM)

-DNAJA2

-GAPDH

Compound 8j (50 μM)+- -- -+ +--

Dnaja2_

KO1

Dnaja2_

KO2

50-kDa

-CASP150-37-

A B C

GFP_

sg1

Dnaja2 K

O1

Dnaja2 K

O20

20

40

60

80

100

LDH

rele

ase

(%)

DMSOVbP8j

* *

******

******

NS NS

GFP_

sg1

Dnaja2 K

O1

Dnaja2 K

O20

20

40

60

80

100

LDH

rele

ase

(%)

VehicleLT

******

*** ***

12

Fig. S3. Actr5–/– RAW 264.7 cells are resistant to VbP. (A) Evaluation of ACTR5 protein loss after treatment of RAW 264.7 cells with sgRNAs targeting Actr5 by immunoblotting. Treatment of RAW 264.7 cells with sgRNAs targeting Actr5, a gene that encodes a core component of the INO80 complex, did not result in significant ACTR5 protein loss. (B) Evaluation of ACTR5, INO80B, and ACTR8 protein levels by immunoblotting after treatment of RAW 264.7 cells in A with VbP (10 µM) for 1 week. This selection resulted in the outgrowth of cells with nearly complete ACTR5 protein loss, consistent with ACTR5 deficiency engendering resistance to VbP. We observed loss of other INO80 subunits in Actr5–/– cells, indicating that loss of one INO80 subunit affects the stability or expression of the other subunits. (C) Cell proliferation time-course of the control and Actr5 knockout cells from B as measured by CellTiter-Glo. Actr5–/– RAW 264.7 cells proliferated more slowly than control cells, accounting for the lack of protein depletion prior to VbP selection. Data are means ± SEM of ten biological replicates. (D,E) RAW 264.7 macrophages from B were treated with VbP (24 h) (D) or LT (3 h) (E) before cytotoxicity was determined by LDH release. Data are means ± SEM of four biological replicates. ***p < 0.001 by two-sided Student’s t-test. Figures (C-E) are representative of three or more independent experiments.

GFP_

sg5

37-

GFP_

sg4

Actr5

_sg3

Actr5

_sg2

75-kDa

-GAPDH

-ACTR5

GFP_

sg4

75-kDa

-ACTR5

Actr5

_sg3

Actr5

_sg2

+- + VbP selection

-GAPDH37--CASP1

-INO80B37-

-ACTR875-

50-

A B

D E

0 20 40 60 800

500

1000

1500

2000

2500

Time (h)

CTG

sig

nal (

% o

f T0) Control

Actr5_sg2

Actr5_sg3

C

Contro

l

Actr5_sg2

Actr5_sg3

0

20

40

60

80

100

LDH

Rel

ease

(%)

BSALT (1 µg/mL)

Contro

l

Actr5_sg2

Actr5_sg3

0

20

40

60

80

100

LDH

Rel

ease

(%)

DMSOVbP (2 µM)

******

13

Fig. S4. LT induces more loss of the NLRP1B full-length protein than the C-terminal fragment. (A) Control (Cas9 only) or Ubr2 KO RAW 264.7 cells were treated with LT (1 µg/mL) for 3 h before lysates were evaluated by immunoblotting. Data are from three biological replicates for each cell line. (B) The band intensities in A were quantified using ImageJ, and the ratio of the C-terminal fragment to the full-length protein was determined. For each cell line, the ratios were normalized to a value of 1 for the vehicle control samples. NS, not significant, ***p < 0.001 by two-sided Student’s t-test.

150-

100--NLRP1B FL

25- -NLRP1B C-term

LT - + - + - + - + - + - + - + - + - +

#1 #2 #3Replicate #1 #2 #3 #1 #2 #3

Ubr2 KO1 Ubr2 KO2

37- -GAPDH

Control

A

B

Control Ubr2KO1Ubr2KO2

0

1

2

3

4

5

6

Rel

ativ

e R

atio

of C

-term

to F

L

ControlLT (3 h)

***

NS NS

14

Fig. S5. Bortezomib rescues VbP-induced degradation of human CARD8. HEK 293T cells stably expressing human caspase-1 and gasdermin D were transiently transfected with a construct encoding CARD8. After 24 h, cells were treated with DMSO or bortezomib for 30 m before the addition of VbP. After 6 h, cell death was assessed by an LDH assay and protein levels were evaluated by immunoblotting. The immunoblots shown are representative of three independent experiments. LDH data are means ± SEM of three biological replicates. ***p < 0.001 by two-sided Student’s t-test. The immunoblot is representative of two independent experiments.

α-CARD8-C

-GAPDH

α-GSDMD

––

+–

++ 20 µM Bortezomib

10 µM VbP

-CARD8-C

-CARD8-FL

-GSDMD FL

-GSDMD CL

-CARD8-N

-CARD8-FL

α-CARD8-N

37-

50-

kDa

37-

25-

50-

37-

50-

0

10

20

30

40

LDH

rele

ase

(%)

******

15

Fig. S6. N-end rule genes are not involved in VbP-induced pyroptosis. (A,B) RAW 264.7 cells with the indicated genotypes were treated with VbP (10 µM) for 6 h before lysates were evaluated by immunoblotting (A) and supernatants were evaluated for LDH release (B). The immunoblots shown are representative of two independent experiments. In B, data are means ± SEM of three biological replicates. Data are representative of three independent experiments.

VbP (6h) - + - + - + - + - +

37-25-

100-

150-kDa-NLRP1B FL

-NLRP1B C-term

-GAPDH

A

B

α-NLRP1B

Control Uba6KOUbr2KO1

Ubr2KO2

Ubr4KO

0

20

40

60

80

100

LDH

Rel

ease

(%)

DMSOVal-boroPro (6 h)

Control Uba6KOUbr2KO1

Ubr2KO2

Ubr4KO

16

Fig. S7. LT activates L45M NLRP1B. (A,B) HEK 293T cells were transiently transfected with plasmids encoding NLRP1B WT or L45M containing N-terminal V5-GFP tags. After 24 h, lysates were harvested and treated with LF (10 ng/mL). Aliquots were removed and quenched at the indicated times. LF cleaves both the WT and mutant protein. (C,D) HEK 293T cells stably expressing mCASP1 and mGSDMD were transiently transfected with construct encoding wild-type NLRP1B or L45M mutant NLRP1B. After 24 h, LT (1 µg/mL) was added to the indicated samples, which were then incubated for an additional 6 h before cell viability was evaluated by LDH release (C) and expression was evaluated by immunoblotting (D). Data are means ± SEM of three biological replicates. ***p < 0.001, **p < 0.01 by two-sided Student’s t-test. Data are representative of two independent experiments.

WT L45MLT+

α-NLRP1B

-GAPDH

Mock-

-NLRP1B FL

-NLRP1B C-term

150-

25-

37-

kDa

*****

NLRP1B WT

NLRP1B L45M

Mock

Time (min)

α-V5150-

37-

37-

-GAPDH

-NLRP1B FL

-LF cleaved NLRP1B

-NLRP1B N-term

kDa

150-

37-

37-

A

B

C

-GAPDH

-NLRP1B FL

-LF cleaved NLRP1B

-NLRP1B N-term

0 15 30 45 60 90 120Mock

Time (min)0 15 30 45 60 90 120

α-V5

0

5

10

15

20

LDH

Rel

ease

(%)

BSALT

WT L45MMock

D

+- +-

17

Fig. S8. N-end rule inhibitors do not block VbP-induced NLRP1B pyroptosis. (A) RAW 264.7 cells were treated with bestatin methyl ester (Me-Bs, 10 µM) or L-Phe-NH2 (1 mM) for 30 min before the addition of VbP (2 µM, 6 h). Supernatants were then evaluated for LDH release. Data are means ± SEM of three biological replicates. ***p < 0.001 by two-sided Student’s t-test. The vehicle controls are the same as Figure 3I. Data are representative of three independent experiments. (B) RAW 264.7 cells were treated with bestatin methyl ester (Me-Bs, 10 µM), L-Phe-NH2 (1 mM), or bortezomib (1 µM) for 30 min before the addition of VbP (2 µM, 6 h). Lysates were then evaluated by immunoblotting. Data are representative of more than five independent experiments. (C) C57BL/6J mice were dosed intraperitoneally with vehicle (0.1 N HCl), VbP (20 µg/mouse), bestatin (50 mg/kg), bestatin methyl ester (50 mg/kg), or the indicated combinations. After 6 h, levels of serum G-CSF were determined by ELISA. Data are means ± SEM, n = 4 mice/group. **p < 0.01, ***p < 0.001 by two-sided Student’s t-test.

A

VbP

Bort

Me-Bs

-

-

-

-

-

-

-

-

-

++ -

+

+

-

+ +

+

- NLRP1B FL

- NLRP1B C-term

- GAPDH

150-

100-

37-25-

kDa

CB

DMSOVbP

0

20

40

60

80

100

LDH

Rel

ease

(%)

DMSOMe-BsL-Phe-NH2

******

Vehicl

eVbP

Bestat

in

Methyl-

besta

tin

VbP +

besta

tin

VbP +

Me-bes

tatin

0

5,000

10,000

15,000

[G-C

SF] (

pg/m

L)

***

***

**

18

Fig. S9. Aminopeptidase inhibition is synergistic with DPP8/9 inhibition in human THP-1 cells. (A,B) DPP8/9 knockout and control THP-1 cells were treated with the indicated compounds for 48 h before cell viability was assessed by CellTiter-Glo. Bestatin, methyl bestatin, batimastat, and CHR-2797 preferentially killed the DPP8/9 knockout cells (A), whereas actinonin and amastatin did not (B). Data are means ± SEM of three biological replicates. (C) DPP8/9 knockout and control THP-1 cells were treated with the indicated compounds (2 µM, 24 h) before lysates were harvested and PARP cleavage (i.e., apoptosis) and GSDMD cleavage (i.e., pyroptosis) were evaluated by immunoblotting. No PARP cleavage was observed, but the aminopeptidase inhibitors induced GSDMD cleavage selectively in the DPP8/9 knockout cells. VbP only induced GSDMD cleavage in control cells, as expected. (D,E) Control (D) or caspase-1 knockout (E) THP-1 cells were treated with the indicated compounds at the indicated concentrations for 24 h before cell viability was determined by CellTiter-Glo. For each pair of concentrations, we subtracted the predicted Bliss additive effect from the observed inhibition. Values greater than zero indicate synergy. We observed synergy in the THP-1 control cells with bestatin methyl ester and CHR-2797 (D), but not in the VbP-resistant CASP1–/– THP-1 cell line (E). Data are means ± SEM of three biological replicates. Data are representative of three (C) or more than five (A, B, D, E) independent experiments.

-GAPDH

VbP ++ +- - - - - -

Me-

Bs

Me-

Bs

Me-

Bs

CH

R 2

797

CH

R 2

797

CH

R 2

797

Bat

imas

tat

Bat

imas

tat

Bat

imas

tat

- - -

+ + +

GFP_sg1 DPP8/9 KO1

-PARP FL100-

50-37-

-14 -12 -10 -8 -6 -40

50

100

150

log([Bestatin]) M

Viab

ility

(%)

sgGFP DPP8/9 KO1DPP8/9 KO2

-14 -12 -10 -8 -6 -40

50

100

150

log([Methyl bestatin]) M

Viab

ility

(%)

-14 -12 -10 -8 -6 -40

50

100

150

log([Actinonin]) M

Viab

ility

(%)

-14 -12 -10 -8 -6 -40

50

100

150

log([Batimastat]) M

Viab

ility

(%)

-14 -12 -10 -8 -6 -40

50

100

150

log([CHR-2797]) M

Viab

ility

(%)

-14 -12 -10 -8 -6 -40

50

100

150

log([Amastatin]) M

Viab

ility

(%)

A B

IC50 = >60 μMIC50 = 789 nM

IC50 = >60 μMIC50 = 1 nM

IC50 = 40 μMIC50 = 87 pM

IC50 = 97 nMIC50 = 10 pM

IC50 = 1 μM IC50 = 1 μM

IC50 > 60 μMIC50 > 60 μM

sgGFP DPP8/9 KO1DPP8/9 KO2

2040.16

0.03

2

0.00

6

0 0.8

E

-PARP CL

-GSDMD FL

-GSDMD CL37-

C

20

4

0.16

0.032

0

0.8

Val-boroPro (μM)

20

4

0.16

0.032

0

0.8

Bes

tatin

met

hyl e

ster

(μM

)C

HR

279

7 (μ

M)

D

0 0 0 0 0 0 0

2 2 5 4 4 -1 0

2 -1 3 2 2 -3 0

1 1 2 2 1 2 0

0 0 1 1 -1 0 0

0 1 0 1 1 1 0

3 3 3 3 3 3 3

2 -3 19 28 21 12 17

2 -3 28 41 35 23 22

3 13 45 48 41 32 35

2 15 40 50 41 33 31

2 19 38 40 31 21 25

-7 -7 -7 -7 -6 -6 -6

-7 0 14 23 18 -1 1

-7 -5 18 18 13 1 -7

-6 1 19 25 14 8 6

-5 0 7 6 2 -3 -7

-3 0 3 7 2 -5 -10

2040.16

0.03

2

0.00

6

0 0.8

20

4

0.16

0.032

0

0.8

Val-boroPro (μM)

Bes

tatin

met

hyl e

ster

(μM

)

19

Fig. S10. The toxicity of the NLRP1B C-terminus is not affected by N-end rule, DPP8/9, or proteasome inhibition. (A,B) HEK 293T cells stably expressing mouse caspase-1 were transiently transfected with constructs (0.2 µg) encoding the FIIND-CARD fragment (with a C-terminal FLAG tag) of NLRP1B with an S984M start site (A) or fused in frame to ubiquitin to generate the native S984 start site (B). After 24 h, cells were treated with the indicated agents for 6 h before supernatants were evaluated for LDH release. Data are means ± SEM of three biological replicates. These agents had no effect on the toxicity of the FIIND-CARD fragment, indicating that all regulation occurs at the level of or upstream of the full-length NLRP1B protein containing the N-terminus. Data are representative of two independent experiments.

A B

DMSOVbP Bort

ezom

ib

Me-Bes

tatin

L-Phe

-NH 2

LT

37-25-

kDa

-GAPDH

-FLAGmoc

k

NLRP1B (S984M-1233)

37-25-

kDa

-GAPDH

-FLAG

NLRP1B (Ub-S984-1233)

DMSOVbP Bort

ezom

ib

Me-Bes

tatin

L-Phe

-NH 2

LTmock

Mock

DMSOVbP

Bortez

omib

Me-bes

tatin

L-Phe

-NH 2 LT

0

10

20

30

40

50

Mock

DMSOVbP

Bortez

omib

Me-bes

tatin

L-Phe

-NH 2 LT

0

10

20

30

40

50

LDH

Rel

ease

(%)

LDH

Rel

ease

(%)

20

Fig. S11. The NLRP1B neo N-terminus is sufficient to induce UBR2/4-mediated degradation. (A,B) HEK 293T cells were transfected with the indicated siRNAs and incubated for 24 h, and then transfected with NLRP1BM1-60- or Nlrp1L45-60-GFP-FLAG fusion constructs (0.05 µg) for an additional 24 h (L45 of NLRP1B was fused in frame to ubiquitin to generate a protein starting with a leucine residue). GFP levels were then assessed by flow cytometric analysis. In A, representative flow cytometric data from 3 biological replicates is shown. The experiment was performed twice with similar results. The x-axis represents forward scatter and the y-axis represents GFP intensity. In B, the percent GFP+ cells from the flow cytometric analysis were quantified. Data are means ± SEM of three biological replicates. Data are representative of two independent experiments.

A

NLRP1BM1-L60-GFP-FLAG

NLRP1BL45-L60-GFP-FLAG

GFP Negative96.6

GFP Positive2.78

0 50K 100K 150K 200K 250K

FSC-A

0

-10 3

10 3

10 4

10 5

GFP

-A

GFP Negative91.8

GFP Positive7.64

0 50K 100K 150K 200K 250K

FSC-A

0

-10 3

10 3

10 4

10 5

GFP

-AG

FPG

FPGFP Negative

94.5

GFP Positive5.07

0 50K 100K 150K 200K 250K

FSC-A

0

-10 3

10 3

10 4

10 5

GFP

-A

GFP Negative93.8

GFP Positive5.90

0 50K 100K 150K 200K 250K

FSC-A

0

-10 3

10 3

10 4

10 5

GFP

-AG

FPG

FP

GFP Negative91.8

GFP Positive7.64

0 50K 100K 150K 200K 250K

FSC-A

0

-10 3

10 3

10 4

10 5

GFP

-A

GFP Negative93.0

GFP Positive6.58

0 50K 100K 150K 200K 250K

FSC-A

0

-10 3

10 3

10 4

10 5

GFP

-AG

FPG

FP

siGAPDH siUBR2 siUBR2/4

BsiGAPDH

siUBR2siUBR2/4

0

2

4

6

8

10

NLRP1B

M1-L60

-GFP-F

LAG

NLRP1B

L45-L

60

-GFP-F

LAG

GFP

Pos

itive

Cel

ls (%

)

21

Fig. S12. N-end rule inhibitors increase levels of the NLRP1BL45-L60-GFP fusion protein. HEK 293T cells were transfected with the indicated NLRP1BM1-60- or NLRP1BL45-60-GFP-FLAG fusion constructs (0.05 µg) 18 h. Cells were then treated with DMSO, Me-Bs (10 µM), or L-Phe-NH2 (1 mM) for 6 h before GFP levels were then assessed by flow cytometric analysis. In A, representative flow cytometric data from 3 biological replicates is shown. The experiment was performed twice with similar results. The x-axis represents forward scatter and the y-axis represents GFP intensity. In B, the percent GFP positive cells from the flow cytometric analysis of the indicated transfections were quantified. Data are means ± SEM of three biological replicates. L-Phe-NH2 reduced NLRP1BM1-L60-GFP-FLAG expression in control cells, likely due to off-target (non-N-end rule) effects. In C, lysates of similarly treated cells were analyzed by immunoblotting. Data are representative of two independent experiments.

A

B

GFP Negative81.4

GFP Positive16.6

0 50K 100K 150K 200K 250K

FSC-A

0

-10 3

10 3

10 4

10 5

GFP

-A

GFP Negative75.7

GFP Positive22.8

0 50K 100K 150K 200K 250K

FSC-A

0

-10 3

10 3

10 4

10 5

GFP

-AG

FPG

FP

GFP Negative93.7

GFP Positive4.97

0 50K 100K 150K 200K 250K

FSC-A

0

-10 3

10 3

10 4

10 5

GFP

-A

GFP Negative70.5

GFP Positive27.8

0 50K 100K 150K 200K 250K

FSC-A

0

-10 3

10 3

10 4

10 5

GFP

-AG

FPG

FP

GFP Negative79.3

GFP Positive19.3

0 50K 100K 150K 200K 250K

FSC-A

0

-10 3

10 3

10 4

10 5

GFP

-AGFP Negative

70.1

GFP Positive28.3

0 50K 100K 150K 200K 250K

FSC-A

0

-10 3

10 3

10 4

10 5

GFP

-AG

FPG

FP

Me-BestatinDMSO L-Phe-NH2

GFP

Pos

itive

Cel

ls (%

)

DMSOMe-Bs (10 µM)L-Phe-NH2 (1 mM)

37-

37- -GAPDH

C

α-FLAG-NLRP1BM1-L60-GFP-NLRP1BL45-L60-GFP

- - ++ -- Me-Bs (10 μM)

NLRP1BM1-L60

-GFP-FLAGNLRP1BL45-L60

-GFP-FLAG

L-Phe-NH2 (1 mM)- - ++ --

NLRP1BM1-L60-GFP-FLAG

NLRP1BL45-L60-GFP-FLAG

0

10

20

30

NLRP1B

M1-L60

-GFP-F

LAG

NLRP1B

L45-L

60

-GFP-F

LAG

22

Fig. S13. N-end rule inhibition blocks NLRP1BL45-L60-GFP ubiquitination. (A,B) Wild-type (A) or UBA6–/– (B) HEK 293T cells were transfected with the indicated constructs (0.5 µg), incubated for 18 h, and then treated with Me-Bs (10 µM) or L-Phe-NH2 (10 mM) for an additional 6 h. Lysates were then immunoprecipitated using anti-FLAG agarose beads and evaluated by immunoblotting. (C) Confirmation of UBA6 knockout in HEK 293T cells by immunoblotting. Data are representative of three independent experiments.

NLRP1BM

1-L

60 -G

FP-F

LAG

NLRP1BL45-L

60 -G

FP-F

LAG

A B

37-

α-FLAG

37- -GAPDH

input

IP: F

LA

G

α-FLAG

37-

50-

75-

100-

150-

- +- +- -

- + +- --

-NLRP1BM1-L60-GFP

-NLRP1BL45-L60-GFP-Ubn

-NLRP1BL45-L60-GFP

-NLRP1BL45-L60-GFP

-NLRP1BM1-L60-GFP

50-

75-

NLRP1BM

1-L

60 -G

FP-F

LAG

NLRP1BL45-L

60 -G

FP-F

LAG

37-

α-FLAG

37- -GAPDH

input

IP: F

LA

G

α-FLAG

37-

50-

75-

100-

150-

L-Phe-NH2

Me-Bs

- +- +- -

- + +- --

-NLRP1BM1-L60-GFP

-NLRP1BL45-L60-GFP-Ubn

-NLRP1BL45-L60-GFP

-NLRP1BL45-L60-GFP

-NLRP1BM1-L60-GFP

50-

75-

WT HEK 293T cells UBA6-/- HEK 293T cells

Me-Bs (10 μM)

L-Phe-NH2 (10 mM)

Me-Bs (10 μM)

L-Phe-NH2 (10 mM)

37-

100-

150--UBA6

-GAPDH

Control

UBA6-/-

C

23

Fig. S14. Proposed model of NLRP1B activation by VbP. DPP8/9 activity prevents the N-terminal ubiquitination and degradation of the NLRP1B N-terminus.

autoinhibitedNLRP1BCARD C

N

N-terminal degradation

caspase-1

CARD C

CARD p20 p10

VbP

DPP8/9

N-terminal ubiquitination

CARD C

NUb

UbUb

free C-terminus

Caspase-1 association

NLRP1Binflammasome

CARD C

CARD p20 p10

proteasome

24

Table S1. sgRNAs enriched in the VbP screen. sgRNAs were ranked by overall fold enrichment relative to the control. Shown are sgRNAs that rank in the top 25 by overall fold enrichment for which additional sgRNAs targeting the same gene also ranked in the top 500 by overall fold enrichment. The overall RIGER rank of each gene is shown in Data S1.

Rank Gene sgRNA (5ʹ à 3ʹ) Fold Increase 1 Nlrp1b GTGTAGGATGCCACAAATGA 1482 2 Casp1 AGTTTCAACATCTTTCTCCG 1036 4 Nlrp1b TCCTGAGCTCTGTAATCACC 693 5 Nlrp1b CCCCAATCACTAATGCCAGT 665 7 Nlrp1a AGACCTGCAGCTGAATGACC 591 8 Casp1 GCAACAAATGTTTCAGCTGA 560 11 Dnaja2 CTCCGCCGCTGCCTTCCCGT 422 18 Casp1 GTATGGGTACCTGAGGATGA 305 19 Stip1 GCAGAACAAGCCGTCAGACC 293 20 Dnaja2 ATTCTTGCTAAGTTGTAGTT 289 25 Ino80b CCGCTCCTAGCGGGCGTGGA 213

25

Table S2. sgRNAs enriched in the LT screen. sgRNAs were ranked by overall fold enrichment relative to the control. Shown are sgRNAs that rank in the top 25 by overall fold enrichment for which additional sgRNAs targeting the same gene also ranked in the top 500 by overall fold enrichment. The overall RIGER rank of each gene is shown in Data S1.

Rank Gene sgRNA (5ʹ à 3ʹ) Fold Increase 1 Antxr2 CTGGCAGTGTAGCAAATAAC 2077 2 Antxr2 TTACAAGCTTTAGTCCTTCA 1853 3 Antxr2 TGTTTCTCCAACTGGCTTAA 1684 5 Ubr4 GGACTTCATCATTGCTGTAA 726 6 Ubr4 CCCCAGTAAGCCCCGAGCTC 645 8 Ubr2 TGTTCTGCCGAAGAGATCGC 499 9 Ubr2 GGCCATCGACCGCAGTTTGC 481 10 Mettl3 TAGGCACGGGACTATCACTA 435 11 Ube2z AACCCCAACTTCTACCGCAA 428 13 Kcmf1 CGATGCAGTGCATATTAACA 415 15 Ubr2 AGGGCCCCGGCAGTAGATTT 377 18 Ube2z CGACACTGTTGACATGACTA 324 20 Ubr4 CAGCTGCCACCGACTGAAGC 320 21 Atp6ap1 TGGGGCTAGCCCCTTGCATG 287 22 Wdr7 ATTGTCACCTCTGAAATGAG 278 23 Tmem199 TCCTAGGTAAGTGCAGACAA 241 24 Zc3h13 TTATGTTCATGAGTTATCAT 239 25 Zc3h13 AAGACTGGCAGCTGCCTCTA 237

26

Table S3. sgRNA sequences used in this study.

Gene sgRNA no. Sequence (5′ à 3′) Used in complete knockout mActr5 2 GGAGCATAGCTACATTGCCG mActr5 3 TCAAATGATGTCGGAGCTCC

mDnaja2 1 ACGGGAAGGCAGCGGCGGAG RAW 264.7 Dnaja2 KO1 and KO2 GFP 1 GGGCGAGGAGCTGTTCACCG GFP 4 GGAGCGCACCATCTTCTTCA GFP 5 GAAGTTCGAGGGCGACACCC

mUbr2 1 GTGGCCGAGTGTTTAAAGTG RAW 264.7 Ubr2 KO1 and Ubr2/4 KO1 mUbr2 2 CCGTTCTGCCCTTTGTTCGC RAW 264.7 Ubr2 KO2 and Ubr2/4 KO2 hUBR2 1 CTTGACATAAAATCCGGCGA HEK 293T UBR2 KO mUba6 2 CTCTTGATGAAACCACAGAC RAW 264.7 Uba6 KO hUba6 1 CATTTGACTGAGTCTTACAA HEK 293T Uba6 KO mUbr4 1 CAGTTACGGAATGTCGGAGG RAW 264.7 Ubr4 KO, Ubr2/4 KO1, and Ubr2/4 KO2

27

Data S1. Genome-wide CRISPR/Cas9 screen results. RIGER values and overall rank for all genes in both the VbP and LT resistance screens.

Data S2. Classification of hits from genome-wide screens. Enriched genes were classified by their known biological roles, which include pyroptosis execution, anthrax entry, INO80 complex, m6A RNA processing, N-end rule pathway, and protein folding. Genes with RIGER p values < 0.01 are highlighted in yellow.

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