Eitan Wong and Irit Sagi - Weizmann Institute of ScienceSelective molecular control in two different...
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Control 1 0.5 0.25 TAPI Control 5 2.5 1.6 TAPI ADAM17 pro-domain (mM) ADAM17 pro-domain (μM) CHO TNF-α secreting cells TNF-α pg/ml (10 2 ) Activated Macrophages TNF-α pg/ml Naive 4 1 Dexa PBS pro-domain (mg/kg) Macroscopic score pro-domain 4mg/kg pro-domain 1mg/kg Dexamethasone PBS Naive Body weight change (%) Day Naive 4 1 PBS pro-domain (mg/kg) Histology score Eitan Wong and Irit Sagi Weizmann Institute of Science, Rehovot, Israel 1.INTRODUCTION ADAM17 (A Disintegrin A Metallproteinese) or TACE (TNF-α converting enzyme) sheds large variety of soluble and cell surface proteins in vitro and in vivo including TNF-α (Tumor Necrosis Factor), which considered one of the main pro-inflammatory cytokines (1,2). TACE is synthesized as a zymogen, the latency of which is maintained by its inhibitory pro- domain. In vivo, proteolytic processing of the pro-domain transforms the zymogen into a catalytically active enzyme (3). We demonstrated that reconstructing the zymogen form with stabilized form of ADAM17 pro-domain specifically attenuates ADAM17 enzymatic activity in vitro, in situ and in vivo. Selective molecular control in two different models of inflammatory bowl disease (IBD) indicates the relevancy of inhibiting ADAM17 in T helper mediated inflammation but not in mechanical tissue rapture induced IBD. 3.ADAM17 PRO-DOMAIN PREVENTS CELL SURFACE SHADDING OF PRO-TNF-α ADAM17 PD inhibits cell surface shedding of pro-TNF-α in activated mouse macrophages and CHO cells stably transfected with human TNF-α . Thus, exhibiting cross-species inhibitory effect in a dose dependent manner in both cell types. 2.NEWLY DESIGNED ADAM17 PRO-DOMAIN FOLD SELECTIVELY INHIBITS ADAM17 IN VITRO Specifically designed stable fold of human ADAM17 pro-domain (PD) was constructed, over expressed and purified to homogeneity. Isolated ADAM17 PD selectively inhibits ADAM17 in vitro. 7.CONCLUSSIONS • The newly designed ADAM17 PD is a natural based selective inhibitor of ADAM17 • ADAM17 PD reduces physical deterioration in T cell mediated colitis • On going work is focused on improving the molecular design of ADAM17 PD and exploring its biological activity in relevant disease models including autoimmune and cancer pathogenesis Purified recombinant ADAM17 pro-domain ADAM17 pro-domain (nM) MMP9 MT1-MMP MMP7 ADAM17 Catalytic activity (%) Naive pro-domain 1 mg/kg pro–domain 4 mg/kg PBS Naive PBS Dexamethasone pro-domain 4 mg/kg pro-domain 1 mg/kg Control pro-domain pro-domain Control 5.ADAM17 PD IMPROVES MANIFISTATION OF T CELL MEDIATED INFLAMMATION BUT NOT TISSUE RAPTURE INDUCE IBD ADAM17 PD exhibits efficacy in TNBS (T cells mediated) induce IBD mice model. However, ADAM17 PD does not attenuate manifestation in DSS (mechanical tissue rapture) IBD model (data not shown). This is achieved by specific localization of the pro-domain to the inflamed colon as seen in fluorescence imaging studies. pro-domain 4mg/kg pro-domain 1mg/kg Dexamethasone PBS Naive Survival (%) Day Colon/Heart ratio 2 Hours 10 Hours PBS/ pro-domain 100 μg LPS Bleed collection +1.5 hour -1 hour 0 hour Naive 2 1 0.5 PBS pro-domain (mg/kg) TNF-α pg/ml 4.IN VIVO REDUCTION OF SOLUBLE TNF-α IN SPESIS MODEL TNF-α plays a major role as a pro-inflammatory mediator and is elevated in septic shock triggering the initiative overwhelming immune response. Injection of endotoxin (LPS) has been used to mimic the septic response in animal models where TNF-α is elevated in the first hours. ADAM17 PD reduces TNF-α in this sepsis model compared to the PBS control. Haptenize autologous protein T Helper mediated inflammation Activation of acquired immune cells TNBS Mechanical damaged Infiltration of bacterial flora Activation of innate immune cells DSS 6.DISSCUSSION In this work we demonstrated that reintroducing the zymogen form of ADAM17 can specifically inhibit its activity in vitro, in situ and in vivo. Treatment with ADAM17 PD proved to be beneficial in TNBS model but not in DSS model, because of the differences in mechanism of disease induction. TNBS involves activation of the acquired immunity and DSS acts in a direct damage of the epithelium where ADAM17 is essential for regeneration (4). References 1. Black, R.A., et al., A metalloproteinase disintegrin that releases tumour-necrosis factor-alpha from cells. Nature, 1997. 385(6618): p. 729-33. 2. Scheller, J., et al., ADAM17: a molecular switch to control inflammation and tissue regeneration. Trends Immunol, 2011. 32(8): p. 380-7. 3. Schlondorff, J., J.D. Becherer, and C.P. Blobel, Intracellular maturation and localization of the tumour necrosis factor alpha convertase (TACE). Biochem J, 2000. 347 Pt 1: p. 131-8. 4. Chalaris, A., et al., Critical role of the disintegrin metalloprotease ADAM17 for intestinal inflammation and regeneration in mice. J Exp Med, 2010. 207(8): p. 1617-24.
Transcript of Eitan Wong and Irit Sagi - Weizmann Institute of ScienceSelective molecular control in two different...
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Control 10.50.25 TAPIControl 52.51.6 TAPIADAM17 pro-domain (mM) ADAM17 pro-domain (µM)
CHO
TNF
-α s
ecre
ting
cells
TNF-α
pg/
ml (
102 )
Activ
ated
Mac
roph
ages
TNF-α
pg/
ml
Naive 41DexaPBSpro-domain (mg/kg)
Mac
rosc
opic
sco
re
pro-domain 4mg/kgpro-domain 1mg/kgDexamethasonePBSNaive
Body
wei
ght c
hang
e (%
)
Day
Naive 41PBSpro-domain (mg/kg)
Hist
olog
y sc
ore
Eitan Wong and Irit Sagi Weizmann Institute of Science, Rehovot, Israel
1.INTRODUCTION
ADAM17 (A Disintegrin A Metallproteinese) or TACE (TNF-α converting enzyme) sheds large variety of soluble and cell surface proteins in vitro and in vivo including TNF-α (Tumor Necrosis Factor), which considered one of the main pro-inflammatory cytokines (1,2). TACE is synthesized as a zymogen, the latency of which is maintained by its inhibitory pro-domain. In vivo, proteolytic processing of the pro-domain transforms the zymogen into a catalytically active enzyme (3). We demonstrated that reconstructing the zymogen form with stabilized form of ADAM17 pro-domain specifically attenuates ADAM17 enzymatic activity in vitro, in situ and in vivo. Selective molecular control in two different models of inflammatory bowl disease (IBD) indicates the relevancy of inhibiting ADAM17 in T helper mediated inflammation but not in mechanical tissue rapture induced IBD.
3.ADAM17 PRO-DOMAIN PREVENTS CELL SURFACE SHADDING OF PRO-TNF-α
ADAM17 PD inhibits cell surface shedding of pro-TNF-α in activated mouse macrophages and CHO cells stably transfected with human TNF-α . Thus, exhibiting
cross-species inhibitory effect in a dose dependent manner in both cell types.
2.NEWLY DESIGNED ADAM17 PRO-DOMAIN FOLD SELECTIVELY INHIBITS ADAM17 IN VITRO
Specifically designed stable fold of human ADAM17 pro-domain (PD) was constructed, over expressed and purified to homogeneity. Isolated ADAM17 PD selectively inhibits ADAM17 in vitro.
7.CONCLUSSIONS• The newly designed ADAM17 PD is a natural based selective inhibitor of ADAM17• ADAM17 PD reduces physical deterioration in T cell mediated colitis • On going work is focused on improving the molecular design of ADAM17 PD and
exploring its biological activity in relevant disease models including autoimmune and cancer pathogenesis
Purified recombinant
ADAM17 pro-domain
ADAM17 pro-domain (nM)
MMP9MT1-MMPMMP7ADAM17
Cata
lytic
act
ivity
(%)
Naive
pro-domain 1 mg/kg pro–domain 4 mg/kg
PBS
NaivePBS
Dexamethasone
pro-domain 4 mg/kg
pro-domain 1 mg/kg
Control
pro-domain
pro-domainControl
5.ADAM17 PD IMPROVES MANIFISTATION OF T CELL MEDIATED INFLAMMATION BUT NOT TISSUE RAPTURE INDUCE IBD
ADAM17 PD exhibits efficacy in TNBS (T cells mediated) induce IBD mice model. However, ADAM17 PD does not attenuate manifestation in DSS (mechanical tissue rapture) IBD model (data not shown). This is achieved by specific localization of the pro-domain to the inflamed colon as seen in fluorescence imaging studies.
pro-domain 4mg/kgpro-domain 1mg/kgDexamethasonePBSNaive
Surv
ival
(%)
Day
Colo
n/He
art r
atio
2 Hours 10 Hours
PBS/pro-domain
100 µg LPS
Bleed collection +1.5 hour
-1 hour
0 hour
Naive 210.5PBSpro-domain (mg/kg)
TNF-α
pg/
ml
4.IN VIVO REDUCTION OF SOLUBLE TNF-α IN SPESIS MODELTNF-α plays a major role as a pro-inflammatory mediator and is elevated in septic shock triggering the initiative overwhelming immune response. Injection of endotoxin (LPS) has been used to mimic the septic response in animal models where TNF-α is elevated in the first hours. ADAM17 PD reduces TNF-α in this sepsis model compared
to the PBS control.
Haptenize autologous protein
T Helper mediated inflammation
Activation of acquired immune cells
TNBS
Mechanical damaged
Infiltration of bacterial flora
Activation of innate immune cells
DSS
6.DISSCUSSIONIn this work we demonstrated that reintroducing the zymogen form of ADAM17 can specifically inhibit its activity in vitro, in situ and in vivo. Treatment with ADAM17 PD proved to be beneficial in TNBS model but not in DSS model, because of the differences in mechanism of disease induction. TNBS involves activation of the acquired immunity and DSS acts in a direct damage of the epithelium where ADAM17 is essential for regeneration (4).
References1. Black, R.A., et al., A metalloproteinase disintegrin that releases tumour-necrosis factor-alpha from cells. Nature, 1997. 385(6618): p. 729-33.2. Scheller, J., et al., ADAM17: a molecular switch to control inflammation and tissue regeneration. Trends Immunol, 2011. 32(8): p. 380-7.3. Schlondorff, J., J.D. Becherer, and C.P. Blobel, Intracellular maturation and localization of the tumour necrosis factor alpha convertase (TACE). Biochem J,
2000. 347 Pt 1: p. 131-8.4. Chalaris, A., et al., Critical role of the disintegrin metalloprotease ADAM17 for intestinal inflammation and regeneration in mice. J Exp Med, 2010. 207(8): p.
1617-24.
