Upload
others
View
2
Download
0
Embed Size (px)
Citation preview
5 0 2 5 1 2 .5 6 .2 5 3 0 0
0
2 0 0
4 0 0
6 0 0
8 0 0
P a r k inW 4 0 3 A
A u t o -U b 2 0 m in
P a r k inW 4 0 3 A
(n M )
% S
ign
al:
Ba
ck
gr
ou
nd
(r
S-r
B/
rB
)x1
00
%
Development of a Real-Time Homogeneous TR-FRET Ubiquitin
Conjugation and Deconjugation Assay PlatformBy South Bay Bio, 5941 Optical Court, Suite 229, San Jose CA, 95138
Contact [email protected], Tel. (415) 935-3226
Ubiquitin, a highly conserved 76 amino acid protein, is an important post-
translational modifier responsible for regulating a wide variety of cellular functions
including but not limited to protein degradation and recycling, cell cycle control, and
DNA damage repair. Ubiquitination, the modification of proteins by the attachment of
ubiquitin or polyubiquitin chains, is catalyzed by a three step process in which ubiquitin
is activated by an ATP-dependent E1 activating enzyme, transferred to an E2
conjugating enzyme, then finally conjugated to a target substrate by an E3 ubiquitin
ligase. The process is reversed by ubiquitin deconjugating enzymes (DUBs.) Thus far,
assay development within the ubiquitin field has suffered a lack of tools for probing the
activity of the conjugation pathway. Herein, we report the development of a novel
assay platform capable of measuring ubiquitin conjugation and deconjugation in real
time in a homogenous, single step assay, utilizing TR-FRET technology.
TR-FRET (Time Resolved Fluorescence Resonance Energy Transfer) uses the extended
fluorescence emission decay lifetimes typical of rare-earth lanthanides to impart a
short time-delay between FRET donor excitation and emission. This delay provides a
ea s to sepa ate t ue sig al f o sho t-lived background fluorescence and reduce
interference from compound fluorescence and other assay artifacts. Using ubiquitin
labeled with either Europium-Cryptate (donor) or Cyanine5 (acceptor), we demonstrate
ubiquitin conjugation and deconjugation measured homogenously in real time, with
assa s o o l e hi iti g Z’ ≥ 0.8. We sho e a ples of auto-ubiquitination
kinetics of several human recombinant E3 ligases of significant interest: MDM2, Parkin,
ITCH, NEDD4, and XIAP. Additionally, we are able to show the reversal of this process
deu i uiti ati g u i uiti ated MDM2 ith the DUB U“P2, a d o pa e U“P2’s kinetics with this substrate versus Ubiquitin-Rhodamine 110.
Activity of All Types of E3 Ubiquitin Ligases can be Measured in a Real-Time Homogeneous TR-FRET Assay
TR-FRET MDM2 Autoubiquitination: Serial dilutions
of MDM2 from 200nM to 12.5nM mixed with UBA1,
UBE2D3, trf-Ub mix. Reaction was initiated with
addition of Mg-ATP, excited at 304 nm, and emission
detected at 620 nm, and 665 nm.
Endpoint E3 Ubiquitin Ligase Autoubiquitination: Conjugation reactions can also be measured in an endpoint configuration. Optimal incubation time has been selected from real-time experiments
where the rate of conjugation is linear. Setup conditions were similar to a real-time reactions, except 10mM DTT and 2mM EDTA were added to quench the reactions before measurement.
DeconjugationDeconjugating enzymes (DUBs) catalyze the removal of ubiquitin from substrate
proteins. This reversal of the conjugation cascade affects critical events in the cell
proteome such as protein degradation through the proteasome. To date, more than
100 DUBs have been annotated, and many studies have shown their multiple functions
in human disease. Current DUB screening reagents largely consist of c-terminal
derivatives such as ubiquitin rhodamine 110 and AMC. However, a growing need for
more physiologically relevant substrates is emerging as we discover more DUBs with
preferential lysine linkage specificities or multimeric ubiquitin chain requirements
TR-FRET TechRare earth cryptates are macrocyclical structures encasing a rare earth lanthanide
ato . La tha ide io s do ’t e hi it suita le fluo es e e p ope ties o thei o , a d e ui e i o po atio ith o ga i oieties fu tio i g as light-ha esti g antennas to collect and transfer energy by intramolecular non-radiative processes.
Our cryptate is composed of a macrocycle in a cage-shaped assembly composed of
three bipyridine arms, which complex a Eu3+ ion (Eu3+TBP) as shown in the structure
below. Unlike other common flavors of rare earth complexes like chelates, cryptates
are extremely robust and stable structures that show no sensitivity to
photobleaching. This is largely due to cryptates having no dissociation between the
complexed ion and the macrocycle, contrary to chelates, which often exhibit
uncoordinated bonds with solvent. These properties allow cryptates to perform in
stringent conditions, e.g. in presence of strong chelators, of divalent cations (e.g.
Mn2+, Mg2+), extremes of pH, organic solvents, and high
temperatures (PCR or other thermoregulated
processes).
Introduction
TR-FRET MDM2-Ubn Chains Titrated with USP2CD: 50nM
MDM2 was conjugated with trf-Ub chains using UBA1,
UBE2D3, and Mg-ATP for 3 hours. TR-FRET MDM2-Ubn
chains were then digested using titrating amounts of USP2CD,
from 93nM to 11nM. Initial velocities (delta (sec-1)) are
shown plotted as a function of USP2CD concentration.
Increasing the DUB concentration increases the reaction rate
linearly.
TR-FRET MDM2-Ubn Chains & USP2CD Ub-Aldehyde Dose-
Response: 50nM MDM2 pre-conjugated with trf-Ub chains
was digested with 250nM USP2CD along with serial dilutions
of Ub-Aldehyde. Initial rates of each inhibitor concentration
were calculated and normalized relative to the no-inhibition
control. Normalized activity was plotted against the log of
inhibitor concentration, and fit with a 4th parameter agonist
curve yielding an IC50 of 152nM. A similar dose-response
using 500nM Ubiquitin-Rhodamine 110 substituted for trf-
MDM2-Ubn yields similar results.
E3 Ligase Autoubiquitination Measured in an Endpoint TR-FRET Assay
TR-FRET MDM2-Ubn Digests with USP2CD & Measures Ki Values
RING HECT RBR
Summary
TR-FRET XIAP Autoubiquitination: Serial dilutions of
XIAP from 350nM to 43.75nM mixed with UBA1,
UBE2D2, trf-Ub mix. Reaction was initiated with
addition of Mg-ATP, excited at 304 nm, and emission
detected at 620 nm, and 665 nm.
TR-FRET ITCH Autoubiquitination: Serial dilutions of
ITCH from 130nM to 8.125nM mixed with UBA1,
UBE2L3, trf-Ub mix. Reaction was initiated with
addition of Mg-ATP, excited at 304 nm, and emission
detected at 620 nm, and 665 nm.
TR-FRET NEDD4 Autoubiquitination: Serial dilutions
of NEDD4 from 50nM to 3.125nM mixed with UBA1,
UBE2L3, trf-Ub mix. Reaction was initiated with
addition of Mg-ATP, excited at 304 nm, and emission
detected at 620 nm, and 665 nm.
TR-FRET ParkinW403A Autoubiquitination: Serial
dilutions of ParkinW403A from 50nM to 6.25nM mixed
with UBA1, UBE2D3, trf-Ub mix. Reaction was
initiated with addition of Mg-ATP, excited at 304 nm,
and emission detected at 620 nm, and 665 nm.
300nM wt-Parkin shown for comparison.
In this report, we show the capabilities of a novel
TR-FRET based platform for probing the activities
of E3 ubiquitin ligases and deubiquitinating
enzymes in real-time and endpoint systems. This
technology fills a longstanding gap in the ability of
researchers to conduct drug discovery with
simple assays tailored for the ubiquitin
proteasome system. Unlike existing technologies,
our platform does not rely on secondary
detection methods such as antibodies. We
de o st ate this platfo ’s o ust ess ith assa s o o l e hi iti g Z’ ≥0.8, a d sho its adaptability to miniaturized formats, making it
ideal for high-throughput screening experiments.
We further provide a proof-of-concept for the
s ste ’s utilit a d si pli it i aki g Ki
determinations, and demonstrate that the
platform holds its weight against a common DUB
substrate, Ubiquitin Rhodamine 110.
0 2 0 4 0 6 0 8 0 1 0 0
-1 0 0
-5 0
0
U b iq u it in a t e d M D M 2 v s . U S P 2 C D T it r a t io n
U S P 2 C D (n M )
Ra
te (
de
lta
)(s
ec
-1)
References
0 1 5 3 0 4 5 6 0 7 5 9 0 1 0 5 1 2 0 1 3 5 1 5 0
0
1 0 0 0
2 0 0 0
3 0 0 0
M D M 2 A u t o u b iq u it in a t io n
t im e (m in )
% S
ign
al:
Ba
ck
gr
ou
nd
(r
S-r
B/
rB
)x1
00
%
2 0 0 n M
1 0 0 n M
5 0 n M
2 5 n M
1 2 .5 n M
1 5 3 0 4 5 6 0 7 5 9 0 1 0 5 1 2 0 1 3 5 1 5 0
0 .0
0 .5
1 .0
M D M 2 A u t o u b iq u it in a t io n Z '
t im e (m in )
Es
tim
ate
d Z
-fa
cto
r
2 0 0 n M
1 0 0 n M
5 0 n M
2 5 n M
1 2 .5 n M
0 1 5 3 0 4 5 6 0 7 5 9 0 1 0 5 1 2 0 1 3 5 1 5 0
0
2 0 0
4 0 0
6 0 0
8 0 0
1 0 0 0
X IA P A u t o u b iq u it in a t io n
t im e (m in )
% S
ign
al:
Ba
ck
gr
ou
nd
(r
S-r
B/
rB
)x1
00
%
3 5 0 n M
1 7 5 n M
8 7 .5 n M
4 3 .7 5 n M
1 5 3 0 4 5 6 0 7 5 9 0 1 0 5 1 2 0 1 3 5 1 5 0
0 .0
0 .5
1 .0
X IA P A u t o u b iq u it in a t io n Z '
t im e (m in )
Es
tim
ate
d Z
-fa
cto
r
3 5 0 n M
1 7 5 n M
8 7 .5 n M
4 3 .7 5 n M
0 1 5 3 0 4 5 6 0 7 5 9 0 1 0 5 1 2 0 1 3 5 1 5 0
0
5 0 0
1 0 0 0
IT C H A u t o u b iq u it in a t io n
t im e (m in )
% S
ign
al:
Ba
ck
gr
ou
nd
(r
S-r
B/
rB
)x1
00
%
1 3 0 n M
6 5 n M
3 2 .5 n M
1 6 .2 5 n M
8 .1 2 5 n M
1 5 3 0 4 5 6 0 7 5 9 0 1 0 5 1 2 0 1 3 5 1 5 0
0 .0
0 .5
1 .0
IT C H A u t o u b iq u it in a t io n Z '
t im e (m in )
Es
tim
ate
d Z
-fa
cto
r
1 3 0 n M
6 5 n M
3 2 .5 n M
1 6 .2 5 n M
8 .1 2 5 n M
0 1 5 3 0 4 5 6 0 7 5 9 0 1 0 5 1 2 0 1 3 5 1 5 0
0
5 0 0
1 0 0 0
1 5 0 0
P a r k inW 4 0 3 A
A u t o u b iq u it in a t io n
t im e (m in )
% S
ign
al:
Ba
ck
gr
ou
nd
(r
S-r
B/
rB
)x1
00
%
5 0 n M
2 5 n M
1 2 .5 n M
6 .2 5 n M
3 0 0 n M w t P a rk in
1 5 3 0 4 5 6 0 7 5 9 0 1 0 5 1 2 0 1 3 5 1 5 0
0 .0
0 .5
1 .0
P a r k inW 4 0 3 A
A u t o u b iq u it in a t io n Z '
t im e (m in )
Es
tim
ate
d Z
-fa
cto
r
5 0 n M
2 5 n M
1 2 .5 n M
6 .2 5 n M
3 0 0 n M w t P a rk in
0 1 5 3 0 4 5 6 0 7 5 9 0 1 0 5 1 2 0 1 3 5 1 5 0 1 6 5 1 8 0
0
1 0 0 0
2 0 0 0
3 0 0 0
N E D D 4 A u to u b iq u it in a t io n
t im e (m in )
% S
ign
al:
Ba
ck
gr
ou
nd
(r
S-r
B/
rB
)x1
00
%
5 0 n M
2 5 n M
1 2 .5 n M
6 .2 5 n M
3 .1 2 5 n M
1 5 3 0 4 5 6 0 7 5 9 0 1 0 5 1 2 0 1 3 5 1 5 0
0 .0
0 .5
1 .0
N E D D 4 A u t o u b iq u it in a t io n Z '
t im e (m in )
Es
tim
ate
d Z
-fa
cto
r
5 0 n M
2 5 n M
1 2 .5 n M
6 .2 5 n M
3 .1 2 5 n M
2 0 0 1 0 0 5 0 2 5 1 2 .5
0
5 0 0
1 0 0 0
1 5 0 0
2 0 0 0
2 5 0 0
N e d d 4 A u t o -U b 3 0 m in
N E D D 4 (n M )
% S
ign
al:
Ba
ck
gr
ou
nd
(r
S-r
B/
rB
)x1
00
%
2 0 0 1 0 0 5 0 2 5 1 2 .5
0
5 0 0
1 0 0 0
1 5 0 0
2 0 0 0
M D M 2 A u t o -U b 3 0 m in
M D M 2 (n M )
% S
ign
al:
Ba
ck
gr
ou
nd
(r
S-r
B/
rB
)x1
00
%
wt
Pa
rkin
1 3 0 6 5 3 2 .5 1 6 .2 5 8 .1 2 5
0
2 0 0
4 0 0
6 0 0
8 0 0
1 0 0 0
IT C H A u t o -U b 3 0 m in
IT C H (n M )
% S
ign
al:
Ba
ck
gr
ou
nd
(r
S-r
B/
rB
)x1
00
%
3 5 0 1 7 5 8 7 .5 4 3 .7 5
0
2 0 0
4 0 0
6 0 0
8 0 0
X IA P A u t o -U b 1 5 m in
X IA P (n M )
% S
ign
al:
Ba
ck
gr
ou
nd
(r
S-r
B/
rB
)x1
00
%
0 .0 0 1 0 .0 1 0 .1 1 1 0
0 .0
0 .5
1 .0
U b -A ld e h y d e T it r a t io n & 2 5 0 n M U S P 2 C D
U b iq u it in -A ld e h y d e (M )
No
rm
ali
ze
d U
SP
2c
d A
cti
vit
y
IC 5 0 = 0 .1 5 2M
M D M 2 -U B n
U B - R H 1 1 0
IC 5 0 = 0 .1 7 5M
Amerik, A. Y., & Hochstrasser, M. (2004). Mechanism and
function of deubiquitinating enzymes. Biochimica et
Biophysica Acta (BBA)-Molecular Cell Research, 1695(1),
189-207.
Koyano, F., Okatsu, K., Kosako, H., Tamura, Y., Go, E.,
Kimura, M., ... & Endo, T. (2014). Ubiquitin is
phosphorylated by PINK1 to activate parkin. Nature,
510(7503), 162.
Magennis, S. W., Parsons, S., Pikramenou, Z., Corval, A.,
& Woollins, J. D. (1999). Imidodiphosphinate ligands as
antenna units in luminescent lanthanide
complexes. Chemical communications, (1), 61-62.
Zheng, N., & Shabek, N. (2017). Ubiquitin Ligases:
Structure, Function, and Regulation. Annual Review of
Biochemistry, (0).
Powered by TCPDF (www.tcpdf.org)Powered by TCPDF (www.tcpdf.org)Powered by TCPDF (www.tcpdf.org)Powered by TCPDF (www.tcpdf.org)