umass dartmouth botulinum research center introduction symposium insights into the mechanism of...
TRANSCRIPT
UMass Dartmouth Botulinum Research Center Introduction Symposium
Insights into the mechanism of BoNT/A neuronal persistence and avenues for novel therapies
George A. Oyler MD, PhDFriday August 24, 2007
Proposed Mechanisms of Persistence
• Cleavage product of SNAP25 by BoNT/A is stable and acts as dominant negative for synaptic transmission. This requires cleavage products from different serotypes to have different recycling time.
• The catalytic subunit is stable and persists in an active form. This requires the different serotypes to have different stability.
• Differential compartmentalization of the catalytic subunits of different serotypes:
YFP-BoNT/A LC is trafficked through multiple vesicle compartments in neuronal cells
GFP-BoNT/A LC is trafficked in a polarized fashion and accumulates in specific sites of neuronal cells
YFP-BoNT/E LC is also trafficked to plasma membrane
YFP-LCE RFP-LCA Merged
N18 neuroblastoma
Differential compartmentalization alone cannot account for differences in persistence
anti-actin
anti-GFP
CHX: 0 1 2 4 6 8 0 1 2 4 6 8
YFP-LCE YFP-LCA
BoNT/A and /E LC stability in SH-SY5Y cells
Ubiquitin proteasome system
250
100
75
50
25
IB: anti-HAIP: anti-GFPIB: anti-GFP
IP: anti-GFP
YF
P
YF
P-L
CE
YF
P-L
CA
YF
P
YF
P-L
CE
YF
P-L
CA
+ HA-Ub
Ubin YFP-BoNT LC
-Ubi-YFP
YFP-LC
YFP
-
LC/A
YFP
-
LC/E
YFP
-
LC/A
YFP
-
LC/E
YFP-BoNT/E LC is ubiquitinated more extensively than YFP-BoNT/A LC in N18 cells
Myc-LC/A Myc-LC/E
HectD2 TRIM63
TRIP12 Cbl-b
E4A
TEB4
Triad3
DZIP3
ProteasomeComplex
Degraded BoNT lc
BoNT LC
Ub
Ub
UbUb
ubiquitination
Proteasomerecognition
slow BoNT degradation
Natural proteasomal turnover of BoNT LC
Designer E3 ligases that target toxins for proteasome degradation
Ub
BoNT lc
UbUb
UbTarget bindin
g domai
n
Ubiquitin E3-ligase
Cellular E3 ligase
Target bindin
g domai
n
Ubiquitin E3-ligase
“slow”
Ub
BoNT lc
UbUb
UbUb
Ub
Degraded BoNT lc
Therapeutic fusion protein
BoNT LC
Ub
Ub
UbUb
Designer E3 ligases that target toxins for proteasome degradation
Enhanced proteasomal turnover of BoNT LC
UbUbUb
Ub
“Designer E3 ligase”
“fast”
ProteasomeComplex
Ub
BoNT lc
UbUb
Ub
E3-ligase
LC bindingagent
E3-ligase
LC bindingagent
BoNT lcubiquitination
Proteasomerecognition
Accelerated BoNT
degradationUb
Ub
Antidotes that accelerate turnover of intraneuronal BoNT LC
Background:
• The concept of targeted proteolysis of cellular proteins has been demonstrated several times in the literature.
• SNAP25/nc based “proof of concept” for a designer E3-ligase strategy.
• For potential therapeutic applications, we are currently developing:
1. Camelid antibodies as more effective LC targeting domains.
2. Optimal E3-ligase domain (e.g. F-box proteins).
3. Neuronal delivery vehicle.
XIAP RING is Catalytic E3 domain
SNAP25 replaces XIAP BIR1-3 domains and recognizes BoNT as substrate for ubiquitination
SNAP-25/NC
BoNT/A and E noncleavable C-terminus of SNAP25
BIR1 BIR2 BIR3
CC
C
C
C
H
C CZn
Zn
XIAP BIR1-3 domains recognizes and binds caspase substrate for ubiquitination
CC
C
C
C
H
C CZn
Zn
SNAP-25/NC-RING
cells alone
SNAP-25/NC Control
SNAP-25/NC-RING + proteasome inhibitor (MG132)
Rel
ativ
e am
ou
nt
of
35S
la
bel
led
YF
P-L
C
Time (hours)
5 10 150 20 25
SNAP-25/NC-RING “designer E3 ligase” substantially accelerates proteasome-mediated degradation of recombinant BoNT/A in transfected neurons
Designer E3 ligase accelerates BoNT/A LC turnover in N18 cells
Spinelli S, Desmyter A, Frenken L, Verrips T, Tegoni M, Cambillau C. Domain swapping of a llama VHH. FEBS Lett. 2004;564(1- 2):35- 40.
Camelid VHH forms a compact well-folding single peptide structure
Background:
• VH domains of camelid HcAbs (VHHs) are easy to produce as recombinant proteins in E. coli and have excellent hydrodynamic properties.
• These antibodies are also generally superior for enzyme neutralization as they bind better into “pockets” such as found in enzyme active sites.
Progress:
• We hyper-immunized two alpacas in New Zealand with A-LC and prepared a VHH phage display library.
• We obtained five unique A-LC binding positives screening at high stringency, three with particularly high apparent affinity.
VHHs as targeting domains
2.5e6 6.43216080040002e410e45e5
ELISA vs BoNT/A LCSDS-PAGE (Coomassie)
A6 E3 D4 G6 B8VHH ELISA on BoNT/A LC
0
0.5
1
1.5
2
2.5
3
3.5
300 60 12.5 2.5 0.5 0.1 0.02
nM VHH
Ab
sorb
ance
A6
E3
D4
G6
B8
Series1
Series2
Series3
Series4
Series5
A6
E3
D4
G6
B8
350 ng each
SDS-PAGE (Coomassie)
A6 E3 D4 G6 B8
350 ng each
VHH-B8 selected as having the highest affinity for BoNT/A LC
Elisa analysis of Anti-BoNT/A Lc VHH clones
GST-VHH-B8 (ug)
YF
P-S
NA
P25
-CF
P
clea
vag
e ac
tivi
ty (
%)
25
50
75
100
VHH-B8 was expressed as a GST fusion protein (GST-VHH-B8). Assays were conducted with 0.2 ug BoNT/A LC in 100 ul reaction volume (25 nM), 0.5 ug YFP-SNAP25-CFP substrate (80 nM), and increasing concentrations of GST-VHH (B8). Inhibition of BoNT/A LC activity by GST-VHH (B8) was near stoichiometric.
0.10 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.90
100% inhibition of 2.5 pM of BoNT/A LC by 0.4 ug, or ~10 pM, of VHH-B8
GST-VHH-B8 potently inhibits BoNT/A Lc
YFP Channel CFP Channel
+ YFP-VHH-RING
- CFP-BoNT/A LC
+ YFP-VHH-RING
+ CFP-BoNT/A LC
- YFP-VHH-RING
+ CFP-BoNT/A LC
Anti-A-LC VHH co-localizes with A-LC in cells
Anti-A-LC VHH localizes to cytosol in transfected Neuro2a
cells.
When co-expressed with BoNT/A LC, the VHH localizes with A-
LC at the plasma membrane.
Western Blot for Steady State level of CFP-BoNT/A LC with YFP-VHH-RING Designer ligases
-BoNT/A LC
Con
trol (
Y-SN
AP25
-C)
YFP-
B8-R
ING
YFP-
D4-
RIN
G
N2a cells Expressing Yes-SNAP25-Cer FRET Indicator
YesFP
CerFP FRETYesFP
CerFPSNAP25 (1-206)
FRET ratio changes from 1.3 to 0.60 over 24 hr treatment with 10 nM BoNT/A in media
Cer BoNT LC Cer BoNT LC
Y B8 onlyCer BoNT LC
Y B8 RingCer BoNT LC
Y B8 TrCP
VHH-B8 inhibits A-LC co-expressed in cells
Transfected BoNT/A LC activity in N2a cell lysates is inhibited when co-transfected with VHH-B8 constructions using YFP/SNAP25/CFP
FRET reduction assay
TrCP designer ligases themselves turnover rapidly
Anti XFP 1:5000
YFP/VHH-B8/TrCP
M - 4 hr o/n
MG135 treatment
Inhibition of proteasomes with MG135 stabilizes TrCP fusion
protein and leads to accumulation of poly-ubiquitinated forms
75
50
37
100
150
25
250
1: YFP-B8 +/A24+24
2: YFP-B8 +/control
3: Indicator only +/A24+24
4: YFP-VHH B8-Trcp +/A24+24
5: YFP-VHH B8-Trcp+/control
6:YFP-VHH B8-Trcp +/A24
7: Indicator only +/A24
8: Indicator only +/control
9: YFP-VHH B8-RING+/A24+24
10: YFP-VHH B8-RING +/control
11: No transfection +/A24+24
12: No transfection +/control
1 2 3 4 5 6 7 8 M 9 10 11 12
Jun. 11th-15th.2007
Anti XFP 1:5000
Anti SNAP 1:5000
1 2 3 4 5 6 7 8 M 9 10 11 12
YFP-B8
NC YFP-SNAP25-CFP
C YFP-SNAP25-CFP
YFP-VHH B8-RING
VHH based designer ligases prevent YFP-SNAP25-CFP cleavage in intoxicated M17 cells.
YFP-B8 YFP-VHH B8-Trcp
YFP-VHH B8-RING
Designer E3 ligases that target toxins for proteasome degradation
Preferred strategy for targeted destruction of BoNT: a smaller, modular “designer E3 ligase”
E3 ligase targeting domain, e.g. minimal TrCP (F-box)
LC bindingagent
VHH-LC targeting domain
Delivery vehicle to neuronal cytosol
E3-ligase
BoNT LC
Note that the targeting domain
can be interchanged to create botulism therapeutics for each serotype once an A-LC prototype has
been developed.
BoNT Lc
BoNT Hc-N
BoNT Hc-C
a. b.
c. d.
BoNT/A Heavy Chain can be used for trafficking cargo to hippocampal organotypic neurons
Conclusions:
1. BoNT/A and /E LC are plasma membrane localized.
2. BoNT/E is degraded much more rapidly than BoNT/A LC in cells.
3. BoNT/E is ubiquitinated and degraded by the proteasome rapidly.
4. Designer E3 ligases can be constructed to accelerate BoNT/A degradation.
5. VHH camelid antibodies have been generated against BoNT/A LC.
6. VHH-based designer E3 ligases are effective in degrading BoNT/A LC.
7. Delivery to intoxicated neurons of VHH-based designer E3 ligases may offer novel post-exposure therapies for BoNT intoxication.
Acknowledgements:Tufts Team:
Chuck Shoemaker PhD
Saul Tzipori DVM PhD
Chueh-Ling Kuo
Jong Beak Park PhD
Ira Herman PhD
University of Maryland:
Paul Fishman MD PhD
Yien Che Tsai PhD (now NCI)
Johns Hopkins:
Daniel Drachman MD
Michael Betenbaugh PhD
Synaptic Research:
George A Oyler MD PhD
James R Oyler
USAMRICD:
Michael Adler PhD
James Eric Keller PhD (now FDA)
Metabiologics:
Michael Goodnough PhD
University of Wisconsin:
Eric Johnson PhD
UMass Dartmouth:
Bal Ram Singh PhD
This work was supported by contract NO1-AI30050 from the National Institutes of Health (NIH) and the National Institute of Allergy and Infectious Diseases (NIAID) and Bioshield NIAID 1R01AI 67504-01 to George A. Oyler MD, PhD