NF-κBシグナル制御におけるHSP70/DNAJA3複合体の機能解析

(1)

NF-κBシグナル制御におけるHSP70/DNAJA3複合体の

機能解析

著者

熊田幸平

学位授与機関

Tohoku University

学位授与番号

11301甲第18894号

URL

http://hdl.handle.net/10097/00129247

(2)

NF-κB

(3)

AGO Argonaute

ARD Ankyrin repeat domain B. bassiana Beauveria bassiana

CERK1 Chitin elicitor receptor kinase 1 c-IAP Cellular inhibitor of apoptosis protein co-IP Coimmunoprecipitation

DD Death domain

DMEM Dulbecco's Modified Eagle's Medium DMSO Dimethyl sulfoxide

DNAJA3 DnaJ homolog subfamily A member 3 Dpt-lacZ Diptericin-lacZ

Drs-GFP Drosomycin-GFP

Dredd Death related ced-3/Nedd-2-like caspase Droj2 DnaJ-like-2

Ecc15 Erwinia carotovora carotovora 15 E. coli Escherichia coli

EDTA Ethylenediaminetetraacetic acid FCS Fetal calf serum

GFP Green fluorescence protein

GNBP Gram-negative bacteria-binding protein GRR Glycine-rich region

GS Gene search system

Gyc76c Gyanylyl cyclase at 76C

HEK293 Human embryonic kidney cells 293 HLH Helix loop helix motif

HOP Hsp70/Hsp90 organizing protein HPD Histidine-proline-aspartate HRP Horse radish peroxidase

HSC70-4 Heat shock protein 70 cognate 4 HSF1 Heat shock factor 1

(4)

HSP Heat shock protein

IgG Immunoglobulin G

IκB Inhibitor of kappa B

IKK IκB kinase

IL Interleukin

IL-1R Interleukin-1 receptor

Imd Immune deficiency

IP Immunoprecipitation

IRAK IL-1 receptor-associated kinase ird Immune response deficient KBD Kinase binding domain

KD Kinase domain

LB Luria-Bertani

LC-MS Liquid chromatography-mass spectrometry LPS Lipopolysaccharide

LZ Leucin-zipper motif

mAb Monoclonal antibody

MyD88 Myeloid differentiation primary gene 88 NAK NF-κB-activating kinase

NB Nutrient broth

NBD NEMO binding domain

NF-κB Nuclear factor kappa B NLS Nuclear localization signal

NP-40 Nonidet P-40

PBS Phosphate buffered saline PES 2-phenylethylene sulfonamide

PEST Proline, glutamimate, serine, threonine-rich sequence PGRP Peptidoglycan recognition proteins

PVDF Polyvinylidene difluoride

RHD Rel homology domain

RIP Receptor-interacting protein kinase RISC RNA-induced silencing complex

(5)

RT Room temperature SDS Sodium dodecyl sulfate siRNA Small interfering RNA

smFRET Single-molecule fluorescence resonance energy transfer

spz Spaetzle

SRD Signal-receiving domain S. saprophyticus Staphylococcus saprophyticus TA1 Transactivation domain 1

TAB TAK1-binding

TAD Transcription activation domain

TAK Transforming growth factor-β-activated kinase TBS Tris buffered saline

TIR Toll-IL-1R

TLR Toll-like receptor TNFα Tumor necrosis factor α

TRADD TNFR1-associated death domain protein TRAF TNF receptor-associated factor

UAS Upstream activation sequence UBD Ubiquitin-binding domain

(6)

1 Droj2 22 Droj2 RNAi 24 Droj2 RNAi 27 Droj2 31 34 HEK293 HSP70/ DNAJA3 NF-κB 37

DNAJA3 HEK293 IKKβ

IκBα NF-κB p65 39

HSP70 HEK293 NF-κB

42

HSP70 HEK293 IKKβ IκBα

NF-κB p65 45

HSP70 HEK293 IκBα NF-κB p65

48

DNAJA3 HSP70 53

HSPA1A HSPA8 HSPA9 HEK293

(7)

HSPA1A HSPA8 HSPA9 IκBα NF-κB p65 60 64 HSPA8 HSC70-4 71 HSC70-4 HSC70-4 73 HSC70-4 75 HSC70-4 77 79 83 90 107 122 123

(8)

2 (1) (2) (3) (4) NF-κB NF-κB

(9)

(5) NF-κB

(5) LPS lipopolysaccharide

flagellin TLR Toll-like receptor

NF-κB TNFα (tumor necrosis factor α)

IL (interleukin)-6

(6, 7) NF-κB

NF-κB (8–10)

NF-κB RelA NF-κB p65 RelB c-Rel NF-κB1

p50 p105 NF-κB2 p52 p100

NF-κB N RHD Rel homology domain

DNA (11–13) NF-κB

NF-κB RelA RelB c-Rel C

TAD Transcription activation domain p50 p52

RelA RelB c-Rel (11–13) Fig. 1 NF-κB

DNA (14) NF-κB

IκB NF-κB IκB

IκB NF-κB NF-κB

(11, 12) IκB N SRD signal-receiving domain

(10)

glutamate-serine- and threonine-rich sequence ARD NF-κB

Fig. 2 IκB IκBα IκBβ IκBε

IκBα RelA p50 NF-κB RHD

Nuclear Localization Signal NLS

NF-κB (11, 12) IκBε RelA

c-Rel/RelA IκBβ RelA/p50

(8, 9, 12, 13) NF-κB p50 p52

p105 p100 C ankyrin repeat

IκBγ IκBδ IκB

(15) IκBγ p50 RelA c-Rel IκBδ RelB

(15, 17, 18) IκB IκBNS BCL3 IκB (19–21) NF-κB IκB ankyrin repeat (22) IκB NF-κB NF-κB

IKK IκB kinase IKK

2 IKKα IKKβ

IKKγ NEMO Fig. 3

IKK

DNA

IKKα IKKβ 50% 70%

N KD (kinase domain) LZ (leucine-zipper motif) HLH (helix loop helix motif) IKKα IKKβ LZ

(11)

HLH KD

(23) IKKα IKKβ C NBD

(NEMO binding domain) NEMO NEMO

HLX1 CC1 HLX2 CC2

LZ (leucine-zipper domain) ZF (zinc-finger

domain) (23) NF-κB NEMO CC2-LZ

ZF K63

IKKα IKKβ NEMO

HLX1 CC1 KBD (kinase binding domain) IKKα

IKKβ NBD IKK

(5) IKK

NF-κB TLR/IL-1R (IL-1 receptor) TNF-α

2 TLR/IL-1R TLR

IL-1R TIR (Toll-IL-1R) MyD88

(myeloid differentiation primary gene 88) MyD88 IRAK (IL-1

receptor-associated kinase) 1 IRAK4 TRAF6 (TNF

receptor-associated factor 6) (24) TRAF6

Ubc13/Uev1A K63

(25) TRAF6 TRAF6 (26), TAK1

(transforming growth factor-β-activated kinase 1)(27) NEMO(28) K63 .

TAK1 TAB (TAK1-binding) 2/3 NEMO

TAK1 IKK

(12)

177 181 IKKα 176 180

(23) IKK IκB SRD

32 36 IκB 48

(14)

IκBα SCFβ-TrCP

IKK IκB RelA (14,

29) NF-κB

TNF-α TNFR1 TNF-α

TRADD (TNFR1-associated death domain protein), TRAF2/5 c-IAP (cellular inhibitor of apoptosis protein) 1/2 RIP1 (receptor-interacting protein kinase 1)

(30). c-IAP1/2 RIP1 K11 K63 (31, 32) IL-1R/TLR TAK1 IKK . NF-κB NF-κB 1 10 TLR IL-1 TNF NF-κB NF-κB IKK

(13)

(12, 33) IKK IKK IKK NF-κB (18) NF-κB TLR Toll (34)

NF-κB Toll Imd 2 (35, 36) (Fig. 4)

Toll (35, 37)

Toll spaezle

(38–40) späzle Toll

dMyD88 Tube Pelle (41, 42)

IκB Cactus

(43, 44) Cactus

NF-κB Dif/Dosal

Drosomycin (45, 46)

Imd PGRP (Peptidoglycan recognition proteins)-LC

PGRP-LE (47–49)

Imd caspase-8 Dredd Death

related ced-3/Nedd-2-like caspase E3 Diap2

E2 (50, 51) Tak1

(14)

IKK (51)

IKK Imd NF-κB Relish

(52) Relish Dredd C Diptericin (53) NF-κB Toll Imd NF-κB TNF-α Fig. 5, 6 NF-κB

Gene Search System GS

GS UAS

upstream activation sequence

(54) UAS GAL4

GAL4 UAS

GAL4 GS

GAL4

heat shock Fig. 7

GAL4 UAS gene

X

(15)

Drosomycin Diptericin lacZ GFP

Diptericin-lacZ Drosomycin-GFP Fig. 8

in vivo

PGRP-LE(55) Toll Gyc76C(56)

1 GS

Droj2 (CG8863) Fig. 9 Droj2

Drosomycin (57) Droj2

DnaJ J domain Zinc finger domain DnaJ C

403 DnaJ

(57) Fig. 9 DnaJ DnaK

ATPase

(58) DnaJ

DnaK (58) DnaJ DnaK

J domain − −

HPD DnaK ATPase

(58). Zinc finger domain CXXCXGXG 4

8 Cys 4 Cys

(16)

(59). DnaJ C DnaJ (58) DnaJ

HSP heat shock protein

70 (60, 61) HSP70 ATP (60) (60) DnaJ HSP70 ATPase HSP70 (61) (Fig. 11) DnaJ HSP70 (60, 61) HSP70 13 DnaJ 49 HSP70 DnaJ (61, 62) DnaJ J domain HSP70 DnaJ HSP70/DnaJ / / NF-κB

HSP90-HOP Hsp70/Hsp90 organizing protein OsCERK1

(17)

(63) NF-κB

HSP90-Cdc37 IKKα/β/γ IKK

TNFα NF-κB

(64) HSP70 IKKγ coiled-coil domain IKKγ

IKK TNFα NF-κB (65) / NF-κB / HSP70/DnaJ NF-κB

(18)

Figure 1 NF-κB

Rel homology domain RHD Transcription activation domain TAD Leucine zipper motif LZ Glycine-rich region GRR Ankyrin repeat

ANK Death domain DD TAD Rel

p52 p50

p100 p105 C NLS ANK

(19)

Figure 2 IκB

Ankyrin repeat ANK proline-glutamate-serine-threonine-rich

sequence (PEST) IκB ANK NF-κB

(20)

Figure 3 IKK

Kinase domain KD Leucine zipper LZ Helix loop helix motif HLH NEMO-binding domain NBD Kinase-binding domain KBD Ubiquitin-binding domain UBD Zinc finger motif ZF

IKKα IKKβ KD IKK

KD

IKKγ/NEMO KBD NBD IKKα/β

(21)

Figure 4 NF-κB

PGRP GNBP

Toll Imd

(22)

Figure 5 Toll TLR

Toll TLR

Toll dMyD88 Pelle Cactus Dif/Dorsal TLR MyD88 IRAK IκB NF-κB

(23)

Figure 6 Imd TNF-α

imd TNF-α

Imd dFADD Dredd

dTAK1 IKK Relish C Relish N RIP FADD

(24)

Figure 7 GAL4/UASシステム

GAL4 は酵母由来の転写活性化因子であり，UAS (upstream activation sequence) に結合してその下流の遺伝子 geneX を転写促進する．本探索系では heat-shock プロモーターの下流に GAL4 が連結されたショウジョウバエを GAL4 系統とし て，また UAS-geneX として GS 系統を用いた．

(25)

Figure 8

Diptericin-lacZ Dpt-lacZ Drosomycin-GFP

Drs-GFP hs GAL4 GS

(26)

Figure 9 GS Droj2

GS Droj2

(27)

Figure 10 Droj2 E.coli DnaJ

Droj2 (CG8863) E.coli DnaJ DnaJ

DnaK DnaK ATPase

- - HPD DnaK

ATPase DnaJ

CXXCXGXG zinc finger DnaK

DnaJ C DnaJ

(28)

21

Nature Reviews Molecular Cell Biology 2010;11(8):579-592

Figure 11 DnaJ HSP70

DnaJ HSP70 DnaJ

(J protein) HSP70 ATPase HSP70

Nature Reviews | Molecular Cell Biology

ATP ADP ADP ATP ATP ATP ADP or 1 2 3 4 5 6 7 Pi NEF J protein Non-native _{client protein}

Native client protein a

HSP70

Adenine

nucleotide-binding cleft Hydrophobic

linker

Peptide-binding cleft

Lid ATPase domain Peptide-binding domain

b

J domain

J proteins drive the multifunctionality of HSP70s. Although it is possible to imagine that the versatility of HSP70 function could be achieved primarily through the amplification and diversification of HSP70 genes during evolution3_{, this does not seem to be the case. The number}

of HSP70s in each cell is rather limited. However, J proteins often far outnumber HSP70s and NEFs in a cellular compartment12,13_{. For example, in mammals there is}

one type of HSP70 in mitochondria and one type in the endoplasmic reticulum (ER), and four and six J proteins, respectively12,13_{. Overall, humans have only 11 HSP70s}

and 13 NEFs, but 41 J proteins (FIG. 3; see Supplementary information S1 (figure)). In addition, and in contrast to HSP70, J proteins show a large degree of sequence and structural divergence (FIG. 3), consistent with the idea that they play a major part in driving the multifunctionality of the HSP70 machinery.

Diversity of J protein structure

The J domain is both the defining domain and the key to a functional interaction with HSP70s as it stimulates their ATPase activity12,13_{. However, many J proteins bear}

little if any sequence or structural similarity outside this domain (FIG. 3; see Supplementary information S2 (figure))and increasing evidence indicates that these non-homologous regions are primary determinants of HSP70 function.

J domain — the common denominator. J proteins, by definition, contain a highly conserved ~70 amino acid signature region, the J domain, which is named after the founding member, Escherichia coli DnaJ (FIG. 4a). Particularly conserved is a His, Pro and Asp tripeptide (HPD), which is in a loop between the two main helices (helix II and helix III). This HPD motif is crucial for J domain function; that is, stimulation of HSP70’s ATPase activity. The exact mechanism of J domain-stimulated ATPase activity and the ensuing conform-ational changes resulting in stabilization of client interaction remain a matter of debate. However, it is established that exposed residues of the J domain form a surface for HSP70 interaction14,15_{. Crucial interactions}

occur with HSP70’s ATPase domain and the adjacent flexible region, which links it to the client protein-binding domain (FIG. 2). These interactions are crucial for transmitting the conformation change necessary for closing the peptide-binding pocket15–20_.

J protein groups, structure and classification. The J pro-tein family has often been referred to as the HSP40 family; but, as most members have a molecular weight that differs greatly from 40 kDa, we refer to them as J proteins throughout this Review. Despite the omni-present J domain, J proteins, as a group, are strikingly dissimilar, with various additional domains (FIG. 3, see Supplementary information S2 (figure)). Historically, J proteins have been divided into three classes (class I, II and III, also known as class A, B and C, respec-tively)13,21-23_{, with class I designation being based on the}

motifs and domains present in E. coli DnaJ. Thus, by def-inition, class I J proteins have an N-terminal J domain, followed by a Gly and Phe-rich region, four repeats of the CxxCxGxG type zinc finger motif and a C-terminal

extension, which is now known to bind client pro-teins24–26_{. This type of C-terminal region is composed}

of two barrel topology domains, C-terminal domain I (CTD I) and CTD II. CTD I has a hydrophobic pocket in which client proteins are thought to bind, as well as a zinc-finger domain extruding from it, which may

Figure 2 | Canonical model of the core HSP70 machinery’s mode of action in

protein folding and HSP70 structure. a | The mode of action of the heat shock 70 kDa

protein (HSP70) core machinery, based on in vitro refolding studies of denatured proteins. J proteins bind to client proteins through their peptide-binding domain (1) and interact with HSP70–ATP through their J domain (2). The client rapidly, but transiently,

interacts with the ‘open’ peptide-binding site of HSP70. ATP hydrolysis is stimulated by both the J domain and client, causing a conformational change in HSP70 that closes the helical lid over the cleft and stabilizes the client interaction, and the J protein then leaves the complex (3). A nucleotide exchange factor (NEF), which has a higher affinity for HSP70–ADP than HSP70–ATP, binds HSP70 (4). The ADP then dissociates through

distortion of the ATP-binding domain (5), after which ATP binds to HSP70 (6). The client is released because of its low affinity for HSP70–ATP (7). ATP binding to HSP70 is favoured as cellular ATP concentrations are typically much higher than those of ADP. If the native state of the client is not attained on release, the J protein rebinds to its exposed

hydrophobic regions and the cycle begins again. b | The structure of HSP70 with ADP bound to the nucleotide-binding domain135_{(protein data bank code}_2KHO_{). The ATPase}

domain and peptide-binding domain are connected by a short, flexible, hydrophobic linker. These domains dock when in the ATP-bound state, which is also thought to displace the lid, allowing easy access and egress of the client protein from the cleft17,136_.

(29)

Droj2

1 Droj2

Droj2

Erwinia carotovora carotovora 15 Ecc15 Beauveria bassiana B. bassiana

(57) Fig. 12

Droj2 RNAi B. bassiana

Droj2 Droj2

Droj2 RNAi Droj2

(30)

Figure 12 Ecc15

Ecc15

1 3 6 12 24 48 72 mRNA

(31)

Droj2 RNAi PGRP-SA PGRP-SD PGRP-LC PGRP-LE (36) Droj2 RNAi GAL4/UAS Droj2

GAL4 c564-GAL4 UAS Droj2

RNA UAS-Droj2 RNAi II

UAS-Droj2 RNAi III Droj2

Droj2 GAL4 UAS GAL80 GAL80ts tub-GAL80ts GAL80ts 18°C GAL4 29°C GAL4 18°C 29°C Droj2 Oregon R yellow

(32)

c564>yellow

Imd NF-κB Relish RelishE20

Toll

spätzle spz spzrm7 Relish

spz

Fig. 13 Droj2 RNAi

Staphylococcus saprophyticus S. saprophyticus

Fig. 13A, D Escherichia coli

E. coli Ecc15 Oregon R yellow

Fig. 13B, C 2 Droj2 RNAi

Droj2 Droj2

(33)

Figure 13 Droj2

Droj2 c564>Droj2 RNAi (II) c564>Droj2 RNAi (III) Oregon R c564>yellow RelishE20, spzrm7

Ecc15 E. coli S. saprophyticus

72 A. B. Ecc15

C. E. coli D. S. saprophyticus

3 100%

(34)

Droj2 RNAi Droj2 NF-κB Droj2 Droj2 RNAi Droj2

total RNA Drosomycin

Diptericin mRNA PCR

Oregon R yellow

c564>yellow

Relish RelishE20

späzle spzrm7

Relish spz

PCR Fig. 14

Ecc15 Diptericin

Oregon R yellow Droj2

E. coli Diptericin Droj2 S. saprophyticus Drosomycin , Droj2 (Fig. 14) Droj2 NF-κB Imd

(35)

(36)

Figure 14 Droj2

Droj2 c564>Droj2

(37)

RelishE20_{, spz}rm7 _{Ecc15 E. coli} _S.

saprophyticus 6

24 total RNA

Diptericin Drosomycin mRNA PCR A.

Ecc15 B. E. coli C. S. saprophyticus Diptericin

Drosomycin rpL32 *p

(38)

Droj2

Droj2 Imd

Droj2 RNAi Droj2 Imd

Droj2 Imd

Imd Imd

Diptericin (66–68)

Imd Droj2 RNAi

Diptericin Imd

Droj2 Diptericin

Droj2

Imd PGRP-LE Imd Dredd Relish

PGRP-LE Imd Dredd Relish

Droj2 Imd

Diptericin Fig. 15

Droj2 Imd Relish

(39)

32 Figure 15 Droj2 RNAi

GAL4 Collagen (Cg)-GAL4 Imd

PGRP-LE imd Dredd Relish

Droj2 RNAi Diptericin PCR

A. PGRP-LE B. imd C. Dredd D. Relish

DNAJA3 Is Required for I!B Phosphorylation—Western blot analysis of human DNAJA3 revealed that flagellin-induced NF-!B accumulation in the nucleus was suppressed in DNAJA3 knockdown cells (Fig. 6, A and B). This finding indicates that DNAJA3 functions upstream of NF-!B nuclear translocation. Analysis of Drosophila Droj2 suggested that Droj2 works genet-ically downstream of Relish or in the regulation of Relish (Fig. 4, A–D). Relish protein contains the N-terminal Rel homology domain and the C-terminal I!B-like ankyrin repeat domain (57). We hypothesized that human Droj2 functions around I!B and NF-!B. Phosphorylated I!B is subjected to protein degra-dation, and phosphorylation of the NF-!B p65 subunit occurs with I!B dissociation and is important for its nuclear translo-cation (13). Western blot analysis for the phosphorylated I!B" and NF-!B p65 revealed that the increase in phosphorylated I!B" and p65 following flagellin stimulation was not observed in DNAJA3 knockdown cells (Fig. 6C). Consistent with this, the decrease in the amount of total I!B" protein following flagellin stimulation was not detected in DNAJA3 knockdown cells. Less I!B signal before stimulation in DNAJA3 knockdown cells

would be due to the fact that I!B is a transcriptional target of NF-!Bs (58). These findings suggest that DNAJA3 is required for the phosphorylation of I!B". IKK is the kinase responsible for NF-!B phosphorylation. Fig. 6D shows that overexpression of the catalytic IKK# subunit induced the phosphorylation of p65 and that phosphorylation was attenuated in DNAJA3 knockdown cells. This finding indicates that DNAJA3 func-tions downstream of IKK.

Discussion

Although the NF-!B signaling pathway is well studied because of its importance in many biologic processes, the com-plete picture of the complex molecular mechanisms of this pathway have not yet been elucidated. In this study, we identi-fied the cochaperone Droj2, which is involved in the Imd path-way, a Drosophila NF-!B pathpath-way, from our genome-wide gain-of-function screening in flies. Epistatic analysis revealed that Droj2 functions downstream of, or parallel to, NF-!B Rel-ish, and a cell culture assay revealed that Droj2 is prerequisite to the nuclear translocation of Relish, suggesting that Droj2 func-FIGURE 4. Droj2 functions genetically downstream of, or parallel to, PGRP-LE, imd, Dredd, and Relish. A–D, quantitative RT-PCR analysis of Diptericin expression following overexpression of PGRP-LE (A), imd (B), Dredd (C), and Relish (D) in larvae carrying UAS-lacZ or UAS-Droj2-RNAi-2. Cg-GAL4 was used to drive the Imd pathway components, and the level of Diptericin expression in each overexpression line was set to 1. The experiments were repeated two to three times and yielded similar results. Data were analyzed using Student’s t test, and values represent the mean ! S.E. of triplicate samples. *, p " 0.05; **, p " 0.01.

Droj2/DNAJA3 Regulates NF-!B Signaling in Drosophila/Humans

at TOHOKU UNIVERSITY on February 9, 2019

http://www.jbc.org/

(40)

Diptericin rpL32 (*p<0.05, **p<0.01; Student’s t test)

(41)

Droj2 Droj2 Droj2 RNAi Ecc15 E. coli NF-κB NF-κB 1 Imd Diptericin Droj2 RNAi

Diptericin Droj2 Imd

Droj2 Imd

Droj2 Imd Relish

Relish C IκB Rel-49

Imd Dredd caspase-8

C (53) RNA

II Drosophila IKK dIKK

Relish N (52, 69) dIKK

Relish Relish DNA

(42)

dIKK Relish Relish Droj2 Relish Relish Droj2 Droj2 Imd Droj2 HSP70 ATPase

RNA Argonaute Ago RNA

(70, 71) RNA Ago

HSP70/HSP40/HSP90 Ago

RNA RISC RNA-induced silencing complex

Relish DNA Dredd dIKK Relish

Droj2

Droj2 Imd

(43)

Drosomycin Drosomycin Imd (72) Droj2 Drosomycin Droj2 DnaJ 36 (62) DnaJ DnaJ Droj2 NF-κB Imd Diptericin NF-κB DnaJ NF-κB

(44)

HEK293 HSP70/ DNAJA3 NF-κB

Droj2 Droj2 Imd

Droj2 J

domain Zinc finger domain

50 DnaJ HSP40

DnaJ

Droj2 NF-κB

HEK293 (Human

Embryonic Kidney cells 293) DnaJ

TLR5 flagellin NF-κB DNAJA3 B7 C6 C8 C27 flagellin NF-κB (73) Droj2 DNAJA3 DNAJA3 HEK293 NF-κB NF-κB IκBα NF-κB p65 DNAJA3 NF-κB (73) DNAJA3 NF-κB IκBα NF-κB p65 NF-κB DNAJA3 NF-κB DNAJA3 HSP70

(45)

NF-κB DNAJA3 HSP70

NF-κB HSP70 HSP70

(46)

DNAJA3 HEK293 IKKβ IκBα NF-κB p65

Droj2 DNAJA3 IκBα

NF-κB p65 flagellin NF-κB

IκBα NF-κB p65 IKK IκBα

NF-κB (74) DNAJA3

IKK IκBα NF-κB p65

DNAJA3 HEK293

IKK 1 IKKβ

immunoprecipitation; IP IKKβ IκBα NF-κB p65

IP input

Non-Target siRNA DNJA3

DNAJA3 input IKKβ IκBα

NF-κB p65 DNAJA3

IKKβ IP

IκBα NF-κB p65 DNAJA3

IκBα NF-κB p65 (Fig. 16, 17)

DNAJA3 NF-κB p65 IKKβ IκBα NF-κB p65

(47)

Figure 16 DNAJA3 IKKβ IκBα NF-κB p65

Non-target DNAJA3 IKKβ

siRNA

(48)

41 Figure 17 Figure 16 ImageJ Input α-tubulin Non-Target siRNA 100% DNAJA3 siRNA

stimulated with the TLR5 ligand ﬂagellin. We found that the NF-

k

B

reporter activity stimulated by ﬂagellin was suppressed by PES in a

concentration-dependent manner (

Fig. 2

A). Next, we analyzed the

IKK

b

/I

k

B

a

/NF-

k

B p65 complex under this condition.

Co-immunoprecipitation experiments revealed that the interactions

of IKK

b

with I

k

B

a

and NF-

k

B p65 were inhibited by PES treatment

(

Fig. 2

B and C). Consequently, the phosphorylation of NF-

k

B p65

induced by ﬂagellin stimulation was suppressed by PES treatment,

whereas the phosphorylation of I

k

B

a

was not impaired (

Fig. 2

D and

E). These ﬁndings suggest that HSP70s regulate NF-

k

B activity

through stabilizing the IKK

b

/I

k

B

a

/NF-

k

B p65 complex.

3.3. DNAJA3 interacted with some HSP70 family proteins

The human HSP70 family has 13 members, and PES inhibits

HSP70 [

17 ,

18 ]. Therefore, we sought to identify the HSP70 family

proteins interacting with DNAJA3 in vivo. For this purpose,

Flag-tagged DNAJA3 protein was transiently expressed in HEK293 cells,

and its interacting proteins were co-immunoprecipitated from the

cell lysate using an anti-Flag antibody (

Fig. 3

A). The interacting

A

B

0 20 40 60 80 100 120 Relativ e Intensity (% of control) Input siRNA NF- p65 0 20 40 60 80 100 120 Relativ e Intensity (% of control) siRNA NF- p65 NF- p65 DNAJA3 -tubulin siRNA Input

Fig. 1. Western blotting analysis of the IKK

b

/I

k

B

a

/NF-

k

B p65 complex under DNAJA3 knockdown. (A) HEK293 cells were transfected with DNAJA3 or control (non-target) siRNA. Cell

lysates (Input) were immunoprecipitated with anti-IKK

b

antibody (IP). Western blotting analysis was performed using anti-IKK

b

, I

k

B

a

, NF-

k

B p65, DNAJA3, and

a

-tubulin

anti-bodies. (B) Quantiﬁcations of the band intensity in A. The relative values normalized by the intensity of control cells are shown. Similar results were obtained from two or three independent experiments (data not shown).

1 3 10 PES 0 20 40 60 80 100 120 NF -re p o rt er ac ti vi ty Flagellin (

A

p-NF- p65 p-NF- p65 p-HSP70 Actin Flagellin 0.1 0.3 1 3 10 PES (

B

0 20 40 60 80 100 120 R ela ti vei n te n si ty ( p-NF- -1 3 10 PES 0.3 0.1 ( Flagellin

D

NF- p65 Actin Input

E

0 20 40 60 80 100 120 Rel ati ve In ten si ty Input NF- p65

C

0 20 40 60 80 100 120 Re lati ve Int ensi ty NF- p65

B-Luc, treated with

PES, and stimulated with ﬂagellin. Values represent the mean ± S.E. of triplicate samples. (B) Western blot analysis of cell lysates after the PES-treatment. The following antibodies were used: anti-IKK

b

, anti-I

k

B

a

, antieNFe

k

B p65, and anti-actin. (C) Quantiﬁcations of the band intensity in B. The relative values normalized by the intensity of control

(DMSO-treated) cells are shown. (D) Cell lysates (Input) were immunoprecipitated with anti-IKK

b

antibody (IP). Western blotting analysis was performed using the following antibodies:

(49)

HSP70 HEK293 NF-κB DnaJ 1 DNAJA3 IKKβ/IκBα/NF-κB p65 NF-κB p65 NF-κB DnaJ HSP70 / DNAJA3 NF-κB HSP70 NF-κB HSP70 NF-κB HSP70 NF-κB Elam-Luc

pRL-TK HEK293 HSP70 pifithrin-µ (PES)

flagellin NF-κB

Elam-Luc

pRL-TK NF-κB

PES HSP70 HSPA1A HSPA1B

(75) HSP70 (76) DMSO flagellin NF-κB PES NF-κB Fig. 18 HSP70 NF-κB

(50)

(51)

Figure 18 HSP70 NF-κB HEK293 2 Elam-Luc pRL-TK HSP70 PES TLR5 flagellin NF-κB NF-κB DMSO 100% PES flagellin

(52)

HSP70 HEK293 IKKβ IκBα NF-κB p65 HSP70 DNAJA3 flagellin NF-κB DNAJA3 IKKβ/IκBα/NF-κB p65 NF-κB HSP70 DNAJA3 IKKβ/IκBα/NF-κB p65

HSP70 HEK293 IKKβ IκBα

NF-κB p65

PES HEK293 IKKβ

IP IKKβ IκBα NF-κB p65

input

DMSO PES

IKKβ IP

IκBα NF-κB p65 PES (Fig. 19,

20)

HSP70 DNAJA3 IKKβ IκBα NF-κB p65

DNAJA3

HSP70 input NF-κB p65

NF-κB p65 DNAJA3

(53)

Figure 19 HSP70 IKKβ IκBα NF-κB p65

DMSO PES IKKβ

(54)

Figure 20 Figure 18 ImageJ Input Actin DMSO 100% PES siRNA

stimulated with the TLR5 ligand ﬂagellin. We found that the NF-

k

B

reporter activity stimulated by ﬂagellin was suppressed by PES in a

concentration-dependent manner (

Fig. 2

A). Next, we analyzed the

IKK

b

/I

k

B

a

/NF-

k

B p65 complex under this condition.

Co-immunoprecipitation experiments revealed that the interactions

of IKK

b

with I

k

B

a

and NF-

k

B p65 were inhibited by PES treatment

(

Fig. 2

B and C). Consequently, the phosphorylation of NF-

k

B p65

induced by ﬂagellin stimulation was suppressed by PES treatment,

whereas the phosphorylation of I

k

B

a

was not impaired (

Fig. 2

D and

E). These ﬁndings suggest that HSP70s regulate NF-

k

B activity

through stabilizing the IKK

b

/I

k

B

a

/NF-

k

B p65 complex.

3.3. DNAJA3 interacted with some HSP70 family proteins

The human HSP70 family has 13 members, and PES inhibits

HSP70 [

17 ,

18 ]. Therefore, we sought to identify the HSP70 family

proteins interacting with DNAJA3 in vivo. For this purpose,

Flag-tagged DNAJA3 protein was transiently expressed in HEK293 cells,

and its interacting proteins were co-immunoprecipitated from the

cell lysate using an anti-Flag antibody (

Fig. 3

A). The interacting

A

B

k

B p65, DNAJA3, and

a

-tubulin anti-bodies. (B) Quantiﬁcations of the band intensity in A. The relative values normalized by the intensity of control cells are shown. Similar results were obtained from two or three independent experiments (data not shown).

A

B

D

NF- p65 Actin Input

B p65, NF-

k

B p65, HSP70, and actin antibodies. (E) Quantiﬁcation of the band intensity in D. The relative values normalized by the intensity of control cells are shown. Similar results were obtained from two or three independent experiments (data not shown).

(55)

HSP70 HEK293 IκBα NF-κB p65 HSP70 DNAJA3 IKKβ/IκBα/NF-κB p65 NF-κB HSP70 IKKβ/IκBα/NF-κB p65 NF-κB IκBα NF-κB p65

PES flagellin HEK293

IκBα NF-κB p65

DMSO flagellin

IκBα NF-κB p65

PES flagellin NF-κB p65

Fig. 21, 22 IκBα

PES Fig. 21 DMSO

IκBα flagellin IκBα

PES IκBα Fig. 21 HSP70 NF-κB p65 NF-κB DNAJA3 IκBα NF-κB p65 (73) Fig. 23 HSP70 NF-κB p65 IKK HSP90 (64, 77) DNAJA3 HSP90 DNAJA3

(56)

(57)

Figure 21 HSP70 IκBα NF-κB p65

PES HEK293 flagellin

IKKα (Ser176) /IKKβ (Ser177) IκBα (Ser32) NF-κB p65 (Ser536)

flagellin

(58)

Figure 22 Figure 21 ImageJ NF-κB p65 (Ser356) NF-κB p65 DMSO flagellin 100% flagellin

stimulated with the TLR5 ligand ﬂagellin. We found that the NF-

k

B

reporter activity stimulated by ﬂagellin was suppressed by PES in a

concentration-dependent manner (

Fig. 2

A). Next, we analyzed the

IKK

b

/I

k

B

a

/NF-

k

B p65 complex under this condition.

Co-immunoprecipitation experiments revealed that the interactions

of IKK

_b

with I

_k

B

_a

and NF-

_k

B p65 were inhibited by PES treatment

(

Fig. 2

B and C). Consequently, the phosphorylation of NF-

k

B p65

induced by ﬂagellin stimulation was suppressed by PES treatment,

whereas the phosphorylation of I

_k

B

_a

was not impaired (

Fig. 2

D and

E). These ﬁndings suggest that HSP70s regulate NF-

k

B activity

through stabilizing the IKK

_b

/I

_k

B

_a

/NF-

_k

B p65 complex.

3.3. DNAJA3 interacted with some HSP70 family proteins

The human HSP70 family has 13 members, and PES inhibits

HSP70 [

17 ,

18 ]. Therefore, we sought to identify the HSP70 family

proteins interacting with DNAJA3 in vivo. For this purpose,

Flag-tagged DNAJA3 protein was transiently expressed in HEK293 cells,

and its interacting proteins were co-immunoprecipitated from the

cell lysate using an anti-Flag antibody (

Fig. 3

A). The interacting

A

B

0 20 40 60 80 100 120

Relativ

e

Intensity

(%

of

control)

Input

siRNA

NF-

p65

0 20 40 60 80 100 120

Relativ

e

Intensity

(%

of

control)

siRNA

NF-

p65

NF- p65 DNAJA3 -tubulin siRNA Input

Fig. 1. Western blotting analysis of the IKK

b

/I

k

B

a

/NF-

k

B p65 complex under DNAJA3 knockdown. (A) HEK293 cells were transfected with DNAJA3 or control (non-target) siRNA. Cell

lysates (Input) were immunoprecipitated with anti-IKK

_b

antibody (IP). Western blotting analysis was performed using anti-IKK

_b

, I

_k

B

_a

, NF-

_k

B p65, DNAJA3, and

_a

-tubulin

anti-bodies. (B) Quantiﬁcations of the band intensity in A. The relative values normalized by the intensity of control cells are shown. Similar results were obtained from two or three

independent experiments (data not shown).

A

B

D

NF- p65 Actin Input

E

C

Fig. 2. Effects of the HSP70 inhibitor on NF-

k

B activity and the IKK

b

/I

k

B

a

/NF-

k

B p65 complex. (A) Luciferase reporter assay in HEK293 cells transfected with NF-

k

B-Luc, treated with

PES, and stimulated with ﬂagellin. Values represent the mean ± S.E. of triplicate samples. (B) Western blot analysis of cell lysates after the PES-treatment. The following antibodies

were used: anti-IKK

b

, anti-I

k

B

a

, antieNFe

k

B p65, and anti-actin. (C) Quantiﬁcations of the band intensity in B. The relative values normalized by the intensity of control

(DMSO-treated) cells are shown. (D) Cell lysates (Input) were immunoprecipitated with anti-IKK

b

antibody (IP). Western blotting analysis was performed using the following antibodies:

anti-phospho-IKK

_a

/

_b

, IKK

_b

, phospho-I

_k

B

_a

, I

_k

B

_a

, phosphoeNFe

_k

B p65, NF-

_k

_{B p65, HSP70, and actin antibodies. (E) Quantiﬁcation of the band intensity in D. The relative values}

normalized by the intensity of control cells are shown. Similar results were obtained from two or three independent experiments (data not shown).

(59)

Figure 23 DNAJA3 HEK293 Flaggelin

IκBα NF-κB p65

DNAJA3 HEK293 flagellin

IκBα (Ser32) NF-κB p65 (Ser536)

(60)

DNAJA3 HSP70 HSP70 DNAJA3 IKKβ/IκBα/NF-κB p65 NF-κB p65 NF-κB HSP70 13 (78) DnaJ (61) DnaJ C DnaJ HSP70 DnaJ 49 DnaJ NF-κB HSP70 DnaJ HEK293 IP LC-MS DNAJA3 HSP70

C FLAG DNAJA3 DNAJA3-FLAG

HEK293 FLAG IP FLAG SDS-PAGE LC−MS input Empty DNAJA3-FLAG Fig. 24 FLAG IP Empty DNAJA3-FLAG Fig. 24

(61)

DNAJA3-FLAG

HSP70

70 kDa * LC-MS

DNAJA3-FLAG co-IP HSP70 HSPA1A HSPA8 HSPA9

ARALAR1/2 Ca

PABPC1 pre-mRNA A

XRCC6 DNA

AIFM1 DDX3X RNA

LMNA A SYNCRIP pre-mRNA

DNAJA3 HSPA1A HSPA8 HSPA9

HSPA1A HSPA8 HSPA9

HSP70 (78) DNAJA3

(79)

HSP70 HSPA1A

HSPA8 (78) HEK293 DNAJA3

flagellin HSP70

(62)

Figure 24 DNAJA3-FLAG FLAG IP SDS-PAGE

C FLAG DNAJA3 HEK293

FLAG IP SDS-PAGE

DNAJA3-FLAG

(63)

HSPA1A HSPA8 HSPA9 HEK293 NF-κB

HSP70 HSPA1A HSPA8

HSPA9 DNAJA3 HSP70

DNAJA3 NF-κB

HSPA1A HSPA8 HSPA9 HEK293

NF-κB Elam-Luc pRL-TK TLR5 flagellin NF-κB HSP70 NF-κB Non-Target siRNA TLR5 MyD88 siRNA Non-Target siRNA flagellin NF-κB

MyD88 siRNA flagellin

NF-κB HSPA1A HSPA8 HSPA9

siRNA NF-κB

Fig. 25 NF-κB

siRNA siRNA

HSP70

(64)

siRNA Fig. 26

siRNA HSPA1A #1 HSPA8 #1 HSPA9 #2 NF-κB

HSP70 flagellin

NF-κB HSPA1A HSPA8

HSPA9 NF-κB

(65)

Figure 25 HSPA1A HSPA8 HSPA9 NF-κB

HSPA1A HSPA8 HSPA9 siRNA HEK293 2

Elam-Luc pRL-TK TLR5

flagellin NF-κB

NF-κB

Non-Target siRNA 100%

(66)

Figure 26 HSPA1A HSPA8 HSPA9

HSPA1A HSPA8 HSPA9 siRNA HEK293 total

RNA PCR mRNA

Non-target siRNA 1

(67)

HSPA1A HSPA8 HSPA9 IκBα NF-κB p65

HSPA1A HSPA8 HSPA9 flagellin NF-κB

HSP70 NF-κB IκBα NF-κB p65

HSPA1A #1 HSPA8 #1

HSPA9 #2 siRNA flagellin

IκBα NF-κB p65

Non-Target siRNA

flagellin IKKα/β NF-κB p65 IκBα IκBα

flagellin MyD88

HSP70 HSPA1A HSPA8

flagellin NF-κB p65

Fig. 27 HSPA9 flagellin

NF-κB p65 IκBα IκBα HSP70 HSP70 DNAJA3 DNAJA3 Non-Target siRNA HSP70 DNAJA3 Fig. 27

(68)

Fig. 27 DNAJA3

HSPA1A HSPA8

NF-κB p65

HSP70 DNAJA3 HSPA1A

(69)

Figure 27 HSPA1A HSPA8 HSPA9 IκBα NF-κB p65

HSPA1A HSPA8 HSPA9 siRNA HEK293 flagellin

IKKα (Ser176) /IKKβ (Ser177) IκBα (Ser32) NF-κB p65 (Ser536)

(70)

(71)

Droj2 DNAJA3 NF-κB IκBα NF-κB p65 NF-κB DNAJA3 HSP70 NF-κB HSP70 flagellin NF-κB NF-κB NF-κB p65 HSP70 NF-κB p65 NF-κB DNAJA3 HSP70 IKKβ IκBα NF-κB p65 DNAJA3 HSP70 IKKβ/IκBα/NF-κB p65 NF-κB p65 (80) C TA1 Ser536 Ser536 TNFα IL-1 LPS (81, 82) IKKα IKKβ (81) HSP70 IKKβ NF-κB p65 NF-κB p65 Ser536 IKK

(72)

(83, 84) HSP70 DNAJA3 IκBα NF-κB p65 (73) HSP70 NF-κB p65 IKK HSP90 (64, 77) DNAJA3 HSP90 (85) DNAJA3 HSP90 IKK DNAJA3 HSP70 HSP90 DNAJA3 HSP70 NF-κB HSP70/DnaJ / (71, 86–88)

HSP70/DNAJA3 IKKβ IKKβ/IκBα/NF-κB

p65 DNAJA3

HSP70 IKKβ IκBα NF-κB p65

Fig.16 Fig.19 2 IKKβ

IκBα NF-κB p65 HSP70 HSP90 -(89) HSP90 HSP90 HSP90

(73)

HSP90 (89)

HSP70 ATP HSP90

(90, 91) HSP70 HSP90

DNAJA3 IKKβ IκBα NF-κB p65

HSP70/DnaJ HSP70 1 HSPA8 DNAJC6 ATPase (92–94) HSP70/DnaJ HSP70 DnaJ HSP70/DnaJ NF-κB HSP70 DnaJ NF-κB HSP70 DnaJ DNAJA3 HSP70 HEK293 HSPA1A

HSPA8 HSPA9 DNAJA3 HSPA1A

HSP70 pH

HSF1 heat shock factor 1 (95, 96)

(74)

HSPA1A

(97, 98) (78) HSPA8

HSP70

(99,

100) HSPA9 HSP70

(101, 102) HSPA1A HSPA8 HSPA9

HSP70 (78) DNAJA3

(79) DNAJA3 HSPA1A HSPA8 HSPA9

HSP70 HSPA1A HSPA8

(78) flagellin HEK293

HSP70 DNAJA3

flagellin NF-κB HSP70 NF-κB NF-κB IκBα NF-κB p65 HSPA1A HSPA8 NF-κB p65 HSP70 NF-κB

HSPA1A HSPA8 IKKβ/IκBα/NF-κB p65

HSPA9 NF-κB p65

(75)

p65 HSPA9 flagellin NF-κB NF-κB (103, 104) flagellin NF-κB HSPA9 NF-κB 1 10 TLR (6, 7) IL-1 TNF (5) NF-κB HSP70/DnaJ NF-κB NF-κB TLR/IL-1R TNF-α IKK flagellin TLR5 NF-κB

NF-κB HSPA1A, HSPA8 DNAJA3

/ NF-κB

(76)

HSP (105) (106–108) (109) (110) (111, 112) (113, 114) LPS HSP70 (115) NF-κB NF-κB HSP NF-κB HSP70 DNAJA3 IKKβ/IκBα/NF-κB p65 NF-κB p65 NF-κB NF-κB

HSPA1A, HSPA8 DNAJA3

/ NF-κB

NF-κB

(77)

(78)

HSPA8 HSC70-4

DNAJA3

HSP70 IKKβ/IκBα/NF-κB p65 NF-κB p65

NF-κB DNAJA3 13

HSP70 HSPA1A HSPA8 HSPA9

HSP70 HSPA1A HSPA8 NF-κB p65 NF-κB HSP70 HSP70 NF-κB HSP70 HSPA8

HSC70-4 Heat shock protein 70 cognate 4 Table 1

HSC70-4 Droj2

(116) HSC70-4 Droj2

(79)

Table 1 HSP70

BLAST https://blast.ncbi.nlm.nih.gov/Blast.cgi HSP70

(80)

HSC70-4 HSC70-4 NF-κB HSPA8 HSC70-4 HSC70-4 RNAi HSC70-4 (117) GAL4

c564-GAL4 UAS HSC70-4 RNA

UAS-HSC70-4 RNAi GAL4

tub-GAL80ts 18°C 29°C HSC70-4 HSC70-4 RNAi total RNA HSC70-4 PCR HSC70-4 2 HSC70-4 Fig. 28

(81)

Figure 28 HSC70-4 HSC70-4 HSC70-4 HSC70-4 PCR HSC70-4 mRNA rp49 mRNA 1

(82)

HSC70-4

HSC70-4 HSC70-4

HSC70-4 RNAi

Ecc15 8

GFP RNAi Imd

NF-κB Relish relishE20

Relish HSC70-4 RNAi 1 #1 Fig. 29 Ecc15 Relish 1 HSC70-4 RNAi #1 #2 HSC70-4 RNAi #1 HSC70-4 Droj2 HSC70-4 RNAi 2 2 HSC70-4

(83)

Figure 29 HSC70-4

HSC70-4

Ecc15 8 .

. Ecc15 3

1.0 *p 0.05; log-rank

(84)

HSC70-4 HSC70-4 Diptericin HSC70-4 Diptericin HSC70-4 RNAi Diptericin HSC70-4

total RNA Diptericin mRNA

PCR GFP Ecc15 Diptericin HSC70-4 1 (#1) Diptericin Fig, 30 HSC70-4 1 (#2) Ecc15 Diptericin (#1) Ecc15 Diptericin HSC70-4 Diptericin HSC70-4 RNAi 2 Diptericin HSC70-4

(85)

Figure 30 HSC70-4 Diptericin

HSC70-4

Ecc15 6 total

RNA Diptericin mRNA PCR

Diptericin rp49 *p 0.05;

(86)

HSP70 HSPA8 HSC70-4 HSC70-4 Ecc15 HSC70-4 Diptericin HSC70-4 Imd HSP70 NF-κB HSC70-4 RNAi 2 Diptericin RNA HSC70-4 (118) Fig. 31 HSC70-4 Droj2 (116) Imd HSC70-4

(87)

Droj2 HSC70-4 Imd HSC70-4 Imd HSC70-4 Imd HSC70-4 Imd Toll HSC70-4 HSP70 HSC70-4 NF-κB HSP70 NF-κB HSP70 HSP70 HSP70 NF-κB HSC70-4 NF-κB HSC70-4 Droj2 NF-κB

(88)

(89)

Figure 31 HSC70-4

(90)

NF-κB

/ NF-κB

Droj2

Droj2 RNAi Droj2

Droj2 RNAi

Diptericin Droj2 Imd

Droj2

Imd Relish

Dredd Relish C dIKK Relish

Relish Droj2 RNAi Relish Relish Droj2 Droj2 Imd Droj2 Drosomycin Droj2 Toll Imd DnaJ 36

(91)

NF-κB DnaJ

DnaJ RNAi

NF-κB

Droj2 J domain Zinc finger domain

50 DnaJ DnaJ Droj2 NF-κB HEK293 DnaJ TLR5 flagellin NF-κB Droj2 DNAJA3 DNAJA3 IκBα NF-κB p65 NF-κB DNAJA3 NF-κB IκBα NF-κB p65 NF-κB DNAJA3 NF-κB DNAJA3 HSP70 NF-κB HSP70 DNAJA3 HSP70 HSP70 flagellin NF-κB NF-κB NF-κB p65 HSP70 NF-κB p65 NF-κB DNAJA3

(92)

HSP70 IKKβ IκBα NF-κB p65 DNAJA3 HSP70 IKKβ/IκBα/NF-κB p65 DNAJA3 HSP70 IKKβ/IκBα/NF-κB p65 1 HSP70 DNAJA3 IKKβ 1

single-molecule fluorescence resonance energy transfer; smFRET

(119, 120) FRET 2

(121) IKKβ

HSP70 DNAJA3 IKKβ IκBα NF-κB p65

HSP70 NF-κB p65 IKKβ/IκBα/NF-κB p65

NF-κB p65 IKK

IKKε NAK NF-κB-activating kinase

(83, 84) HSP70

HSP70

(93)

HSP70 DNAJA3 NF-κB

HSP70/DnaJ HSP70 DnaJ

NF-κB HSP70 DnaJ

DNAJA3 HSP70

HEK293 HSPA1A HSPA8 HSPA9 DNAJA3

HSP70 DNAJA3

DNAJA3 HSP70

NF-κB HSPA1A

HSPA8 NF-κB p65 NF-κB

HSPA1A HSPA8 IKKβ/IκBα/NF-κB p65

HSP70 HSPA9 NF-κB p65 NF-κB HSPA9 NF-κB HSP70 NF-κB HSP70 HSP70 HSPA8 HSP70 HSC70-4 HSC70-4

(94)

Ecc15 Ecc15 Diptericin HSC70-4 Imd HSP70 NF-κB HSC70-4 Droj2 Imd HSC70-4 Droj2 HSC70-4 Imd HSC70-4 Imd HSC70-4 Imd Toll HSC70-4 HSP70 NF-κB HSP70 NF-κB Toll Imd HSP70 HSP70 NF-κB NF-κB HSP70/DnaJ

(95)

flagellin TLR5 NF-κB HSP70/DNAJA3 / NF-κB / HSP70/DnaJ NF-κB HSPA1A/HSPA8 DNAJA3 HSC70-4 Droj2 NF-κB HSP70/DnaJ 1 (Fig. 32) HSP70/DnaJ NF-κB NF-κB NF-κB

(96)

(97)

1. / 10.5% 4% 6.5% 4-0.3% 1% 25°C 18°C 29°C 2. 2-1.

c564-GAL4 tub-GAL80ts UAS-yellow UAS-GFP RNAi

UAS-Droj2 RNAi UAS-HSC70-4 RNAi

Oregon R ( wild type ) RelishE20/ RelishE20 spzrm7/spzrm7

w;c564-GAL4/c564-GAL4;tub-GAL80ts/tub-GAL80ts w;+/+;UAS-Droj2 IR/UAS-Droj2 IR (VDRC #23637) w; UAS-Droj2 IR/UAS-Droj2 IR;+/+ (VDRC #23638) yw;+/+;UAS-yellow/UAS-yellow (Bloomington #3043) w;+/+;UAS-GFP IR/UAS-GFP IR (Bloomington #9383)

(98)

w;Cg-GAL4/GFP-CyO;+/+ Bloomington UAS-Dredd on 3rd chromosome P. Meier UAS-PGRP-LE 13-15-1 on 3rd chromosome UAS-Relish on 2nd chromosome

UAS-Imd on 2nd chromosome

UAS-HSC70-4 RNAi R1 (NIG #4264 R-1) UAS-HSC70-4 RNAi R2 (NIG #4264 R-2)

2-2.

10-15 mL 1.0 mL

2 mL 3,600 g, 5 min, RT

1 mL 2,000 g 5 min RT

1 mL GeneQuant pro GE Healthcare

OD600 OD600≒1

1 1 70 nL

Narishige PC-10 Drummond

Scientific company 3-000-203-G/X Heater Level No.1 70.0 No.2 58.0 Nanoject Ⅱ Drummond Scientific company 3-000-204

OD600

Escherichia coli (K-12) OD600≒1.0 LB 37°C

Erwinia carotovora carotovora 15 OD600≒1.0 LB 30°C

(99)

2-3. 3-7 25 29°C 7 3 1 3 2-4. LB LB plate

LB Broth Miller Nacalai tesque 20068-75 12.5 g MilliQ H2O 500 mL 121°C 20

LB plate LB Broth Miller 5.0 g Agar Nacarai tesque

01028-85 3.0 g MilliQ H2O 200 mL 121°C 20

10 cm plate 10

2-5. NB Nutrient Broth NB plate

Beef extract Oxoid 1.5 g Special Peptone Oxoid 2.5 g MilliQ H2O 500

mL pH 7 121°C 20

NB plate beef extract 0.6 g special peptone 1.0 g MilliQ

H2O 200 mL pH 7 Agar 3.0 g

121°C 20 10 cm 10

(100)

3-1. Trizol total RNA total RNA 10 03961CK28 TRIzol (Invitrogen) 500 µL total RNA TRIzol 500 µL (Nacalai tesque) 150 µL 6,000 rpm 30

Bertin Technologies Precellys 24 5

16,000 g, 15 min, 4°C 300 µL

(Nacalai tesque) 300 µL

RNA 5 (16,000 g, 15 min, 4°C)

DEPC 75% 100% Nacarai

tesque :DEPC =3:1 400 µL (16,000 g, 5 min, 4°C)

75% 400 µL (16,000 g, 5 min, 4°C)

30 µL RNase, DNase-free

RNA RNA 1.5 µL NanoDrop

2000 Thermo scientific OD260 OD260=1

40 mg/mL RNA

3-2. HEK293 total RNA

total RNA Quick-RNA Micro-Prep kit ZYMO RESEARCH

(101)

ZR RNA Buffer Zymo-Spin IC Column 12,000 g 1

min RT Flow-through RNA wash buffer 700 µL

12,000 g 30 sec RT Flow-through

RNA wash buffer 400 µL 12,000 g 30 sec RT Flow-through 12,000 g 2 min RT

wash buffer RNase-free 1.5 mL

DNase/RNase-free water 10 µL 1

20,400 g 30 sec RT RNA

RNA 1.5 µL NanoDrop 2000 OD260

OD260=1 40 mg/ml RNA

3-3. 1st strand cDNA

RNA 1.0 µg oligo (dT)15 primer Promega 1.0 µL RNase, DNase-free

12.5 µL 70°C 10 1

flash

42°C 5 Rever Tra Ace (Toyobo) 1.0

µL 42°C 50 99°C 5

flash −30°C PCR

12.5 µL 5x RT reaction buffer Toyobo 4.0 µL 10 mM dNTPs Promega 2.0 µL RNase inhibitor Promega 0.5 µL

(102)

3-4. Light Cycler Roche Diagnostics PCR

PCR H2O (Roche) 12.4 µL 25 µM MgCl2 (Roche) 1.6 µL 10 µM primer forward reverse 1.0 µL, 10x LC DNA master (Roche) 2.0 µL, 10

cDNA 2.0 µL LightCycler

(400 g 2 sec RT) LightCycler (Roche Diagnostics)

PCR primer

PCR melting

curve

PCR

rp49 95°C, 10 min → ( 95°C, 15 sec → 60°C, 10 sec → 72°C, 8 sec ) × 40 cycles → Melting → 40°C, 30 sec

rpL32 95°C, 10 min → ( 95°C, 15 sec → 60°C, 10 sec → 72°C, 8 sec ) × 40 cycles → Melting → 40°C, 30 sec

Diptericin 95°C, 10 min → ( 95°C, 15 sec → 60°C, 10 sec → 72°C, 10 sec ) × 40 cycles → Melting → 40°C, 30 sec

Drosomycin 95°C, 10 min → ( 95°C, 15 sec → 58°C, 10 sec → 72°C, 8 sec ) × 40 cycles → Melting → 40°C, 30 sec

HSC70-4 95°C, 10 min → ( 95°C, 15 sec → 60°C, 10 sec → 72°C, 8 sec ) × 40 cycles → Melting → 40°C, 30 sec

GAPDH 95°C, 10 min → ( 95°C, 15 sec → 60°C, 10 sec → 72°C, 8 sec ) × 40 cycles → Melting → 40°C, 30 sec

HSPA1A 95°C, 10 min → ( 95°C, 15 sec → 60°C, 10 sec → 72°C, 8 sec ) × 40 cycles → Melting → 40°C, 30 sec

(103)

× 40 cycles → Melting → 40°C, 30 sec

HSPA9 95°C, 10 min → ( 95°C, 15 sec → 60°C, 10 sec → 72°C, 8 sec ) × 40 cycles → Melting → 40°C, 30 sec

Melting ; 95°C→65°C 15 sec→65°C ~95°C

Light Cycler Primer

rpL32 (rp49) -F 5'- AGATCGTGAAGAAGCGCACCAAG -3' rpL32 (rp49) -R 5'- CACCAGGAACTTCTTGAATCCGG -3' Diptericin-F 5'- GTTCACCATTGCCGTCGCCTTAC -3' Diptericin-R 5'- CCCAAGTGCTGTCCATATCCTCC -3' Drosomycin-F 5'- TTGTTCGCCCTCTTCGCTGTCCT -3' Drosomycin-R 5'- GCATCCTTCGCACCAGCACTTCA -3' HSC70-4-F 5'- ACAAGAAGGCTGTTGGAGAGC -3' HSC70-4-R 5'- GGACTTGACCTCAAAGATACCGT -3' GAPDH-F 5’- GCACCGTCAAGGCTGAGAAC -3’ GAPDH-R 5’- TGGTGAAGACGCCAGTGGA -3’ HSPA1A-F 5’-CCTGGAGTCCTACGCCTTCAAC -3’ HSPA1A-R 5’- CTTGACACTTGTCCAGCACCTTC -3’ HSPA8-F 5’- GGAAATTGCAGAAGCCTACCTTG -3’ HSPA8-R 5’- CTTTGGTAGCCTGACGCTGAGA -3’ HSPA9-F 5’- TTAGACTTGTTTCAAGGCGGGATTA -3’ HSPA9-R 5’- TGGCACCTTCGGCATTCTC -3’

(104)

4-1. siRNA 96well

96 Well Cell Culture Plate greiner bio-one HEK293 40-50

100 µL DMEM gibco 10% FCS biowest

2 mM L-glutamine gibco 37°C CO2 5.0

OPTI-MEM gibco 20 µL 1 µM siRNA QIAGEN FlexiTube siRNA 0.4 µL

Lipofectamine RNAiMAX Invitrogen 0.4 µL 10

well 37°C CO2 5.0 48

siRNA

＊ siRNA

AllStars Negative Control siRNA (QIAGEN #1027280) FlexiTube siRNA (QIAGEN)

Hs_MYD88_5 AACTGGAACAGACAAACTATC Hs_DNAJA3_5 CTCCGGCATGGAAACCATCAA Hs_HSPA1A_3 (#1) AGAGATGAATTTATACTGCCA Hs_HSPA1A_9 (#2) TCCGGTTTCTACATGCAGAGA Hs_HSPA8_6 (#1) AAGGACCTAAATTCGTAGCAA Hs_HSPA8_12 (#2) TTAAGCTGCTATAGTAAGTTA Hs_HSPA9_3 (#1) ATGAAGTTGACCCGTGCTCAA Hs_HSPA9_5 (#2) AATTGTATTCTCCGAGTCAGA 4-2. Reporter plasmid

96 Well Cell Culture Plate HEK293 60-80

100 µL DMEM 10% FCS 2 mM L-glutamine 37°C CO2

(105)

Invitrogen 0.2 µL Lipofectamine LTX Invitrogen 0.5 µL

10 96

well plate well 37°C CO2 5.0 18

＊

pELAM-Luciferase 100 ng/well

pRL-TK 20 ng/well Promega

4-3. HSP70

Reporter plasmid 18 HEK293

0.1 0.3 1 3 10 µM pifithrin-µ (PES) Sigma well

37°C CO2 5.0 1

4-4.

FLA-ST Ultrapure InvivoGen 3 ng/mL 96 well plate

well 37°C CO2 5.0 4

4-5. Luciferase

96 well plate well 80 µL Dual-glo Luciferase Buffer Dual-glo Luciferase Substrate Promega Dual-glo Luciferase assay system

70 µL , 10 Molecular

device SpectraMax L-TYA Firefly Luciferase activity

(106)

Dual-glo Luciferase assay system 70 µL 10

Molecular device SpectraMax L-TYA Renilla Luciferase activity

5. IKKβ

5-1. HSP70

100 mm Cell Culture Dish BD Falcon HEK293 100

DMEM 10% FCS 2 mM L-glutamine 37°C

CO2 5.0 10 µM PES 37°C

CO2 5.0 1

5-2. siRNA

100 mm Cell Culture Dish HEK293 40-50

DMEM 10% FCS 2 mM L-glutamine 37°C CO2

5.0 OPTI-MEM 2.0 mL 10 µM siRNA QIAGEN FlexiTube

siRNA 4.0 µL Lipofectamine RNAiMAX 40 µL 10

37°C CO2 5.0 36

siRNA

＊ siRNA

(107)

5-3.

PES siRNA 5.0 mL PBS

SIGMA pH7.4 500 µL cell lysis buffer 25 mM Tris

(SIGMA), 150 mM NaCl ( ), 1% NP-40 ( ), 1 mM

EDTA, Complete Mini (Roche) 1.5 mL Quality

Scientific Plastics 20 15,000 g, 4°C,

10 min 1.5 mL DC Protein Assay Kit (Bio-Rad)

input Flagellin 15

FLA-ST Ultrapure 300 ng/mL 37°C CO2 5.0

15

5-4. IKKβ

5-3 200 µL 2 1.5 mL IKKβ

IKKβ (D30C6) Rabbit mAb (Cell Signaling)

Rabbit (DA1E) mAb IgG XP Isotype Control 2.0 µL 4°C

2 30 µL slurry

Dynabeads M-280 Sheep anti-Rabbit IgG (novex) cell lysis buffer

2 /1

4°C 45 Cell lysis

buffer 3 1x SDS sample buffer 50 mM Tris-HCl

SIGMA 2% SDS 10% glycerol 100 mM

β-mercapto ethanol SIGMA 0.0125 Bromo phenol blue Wako 30 µL

95°C 5 boil

(108)

5-5. SDS-PAGE

10% SDS-PAGE

Mini-PROTEIN III Electrophoresis Cell Bio-Rad 40 mA

Mini-PROTEIN III Electrophoresis Cell Bio-Rad

100 V PVDF GE

Healthcare Hybond-P 1 PVDF

5% /TBST (0.1% Tween20 nacalai tesque

TBS) 1 PVDF

1 1 TBST 5

3 HRP IgG GE Healthcare

5,000 2 1

TBST 5 3 Luminata Forte Western HRP Substrate

MILLIPORE HRP ImageQuant

LAS 4000 mini GE Healthcare 1 2

＊

α-tubulin (ab18251) Rabbit polyclonal Ab Abcam 800 Actin (ab179467) Rabbit mAb Abcam 10,000

Tid-1 (RS13) Mouse mAb Cell Signaling 1,000

IκBα (L35A5) Mouse mAb (Amino-terminal Antigen) Cell Signaling 1,000

NF-κB p65 (D14E12) XP Rabbit mAb Cell Signaling 1,000 IKKβ (D30C6) Rabbit mAb (Cell Signaling, 1,000 )

(109)

＊ 2

ECL Anti-rabbit IgG, Horseradish Peroxidase linked whole antibody (from sheep) (GE Healthcare 5,000 )

ECL Anti-mouse IgG, Horseradish Peroxidase linked whole antibody (from sheep) (GE Healthcare 5,000 )

Mouse Anti-Rabbit IgG (Light-Chain Specific) (D4W3E) mAb (HRP Conjugate) (Cell Signaling 5,000 )

6. DNAJA3 DNAJA3 HSP70

6-1. DNAJA3-FLAG

6 well plate BD Falcon HEK293 60-80

DMEM 10% FCS 2 mM L-glutamine 37°C

CO2 5.0 OPTI-MEM 400 µL

pcDNA3/DNAJA3-FLAG 2.5 µg Plus Reagent 8.0 µL Lipofectamine LTX 10

µL 10

well 37°C CO2 5.0 24

6-2.

1.0 mL PBS 300 µL cell lysis buffer

1.5 mL 20 15,000

g, 4°C, 10 min 1.5 mL DC Protein Assay Kit

(110)

input

6-3. FLAG

6-2 200 µL 1.5 mL 0.1 mg/mL

FLAG FLAG M2 Mouse mAb (Sigma) 2.0 µL 4°C

2 30 µL slurry

Dynabeads M-280 Sheep anti-Mouse IgG (novex) cell lysis buffer

2 /1

4°C 45 Cell lysis buffer

3 300 µg/mL FLAG peptide 40 µL

10 1/3

4x SDS sample buffer 95°C 5 boil IP

6-4. SDS-PAGE

4–15% TGX Bio-Rad

SDS-PAGE Mini-PROTEIN III Electrophoresis Cell

40 mA EzStain Silver Atto

150 mm dish 20 mL 25 mL 5 mL S-1 0.5 mL 10 50 mL 10 3 50 mL S-2 0.5 mL 5 50 mL 30 100 mL S-3 0.5 mL S-4 0.5 mL 50 mL 30

(111)

50 mL

10

50 mL 5 2

1.5 mL

LC-MS

7. HSP70 HSPA1A HSPA8 HSPA9

NF-κB

7-1. HSP70

6 well plate HEK293 100

DMEM 10% FCS 2 mM L-glutamine 37°C CO2 5.0

10 µM PES 37°C CO2

5.0 1

7-2. siRNA RNAi

6 well plate HEK293 40-50

DMEM 10% FCS 2 mM L-glutamine 37°C CO2 5.0 OPTI-MEM 400 µL 10 µM siRNA QIAGEN FlexiTube siRNA

1.0 µL Lipofectamine RNAiMAX 10 µL 10

well 37°C CO2 5.0 36

(112)

siRNA

Hs_MYD88_5 AACTGGAACAGACAAACTATC Hs_HSPA1A_3 (#1) AGAGATGAATTTATACTGCCA Hs_HSPA8_6 (#1) AAGGACCTAAATTCGTAGCAA Hs_HSPA9_5 (#2) AATTGTATTCTCCGAGTCAGA

7-3.

FLA-ST Ultrapure 300 ng/mL 6 well plate well 37°C

CO2 5.0 15 1.0 mL PBS

200 µL cell lysis buffer 1.5 mL

20 15,000 g, 4°C, 10 min

1.5 mL DC Protein Assay Kit

1/3 4x SDS sample buffer

95°C 5 boil

7-4. SDS-PAGE

10% SDS-PAGE

Mini-PROTEIN III Electrophoresis Cell 40 mA

Mini-PROTEIN III Electrophoresis Cell 100 V

PVDF 1

PVDF 5% /TBST

(113)

1 1 4°C TBST 5

3 HRP IgG

5,000 2 1 TBST 5

3 Luminata Forte Western HRP Substrate HRP

ImageQuant LAS 4000 mini

1 2

＊

Tid-1 (RS13) Mouse mAb Cell Signaling 1,000 HSP70 (6B3) Rat mAb Cell Signaling 1,000

IκBα (L35A5) Mouse mAb (Amino-terminal Antigen) Cell Signaling 1,000

Phospho-IκB-α (Ser32) (14D4) Rabbit mAb Cell Signaling 1,000 NF-κB p65 (D14E12) XP Rabbit mAb Cell Signaling 1,000

Phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb Cell Signaling 1,000 Actin (ab179467) Rabbit mAb abcam 10,000

IKKβ (D30C6) Rabbit mAb (Cell Signaling, 1,000 )

Phospho-IKKα (Ser176) / IKKβ (Ser177) (C84E11) Rabbit mAb (Cell Signaling,

1,000 )

＊ 2

ECL Anti-rabbit IgG, Horseradish Peroxidase linked whole antibody (from sheep) (GE Healthcare 5,000 )

ECL Anti-mouse IgG, Horseradish Peroxidase linked whole antibody (from sheep) (GE Healthcare 5,000 )

(114)

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2. Fearon, D. T., and Locksley, R. M. (1996) The instructive role of innate immunity in the acquired immune response. Science (80-. ). 272, 50–54

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5. Napetschnig, J., and Wu, H. (2013) Molecular Basis of NF-κB Signaling. Annu. Rev. Biophys. 42, 443–468

6. Kawai, T., and Akira, S. (2010) The role of pattern-recognition receptors in innate immunity: Update on toll-like receptors. Nat. Immunol. 11, 373–384 7. Akira, S., Uematsu, S., and Takeuchi, O. (2006) Pathogen recognition and innate

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8. Bassères, D. S., and Baldwin, A. S. (2006) Nuclear factor-κB and inhibitor of κB kinase pathways in oncogenic initiation and progression. Oncogene. 25, 6817– 6830

9. Courtois, G., and Gilmore, T. D. (2006) Mutations in the NF-κB signaling pathway: Implications for human disease. Oncogene. 25, 6831–6843 10. Toubi, E., and Shoenfeld, Y. (2004) Toll-like receptors and their role in the

development of autoimmune diseases. Autoimmunity. 37, 183–188

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NF-κBシグナル制御におけるHSP70/DNAJA3複合体の機能解析