Fluorescence Polarization (FP) and Chemiluminescent Based Substrate Alternatives for Analyzing DUB Activity
Nate Russell*, J. Tres Brazell*, Carsten Schwerdtfeger*, Farid El
†
Oualid ,
Huib
†
Ovaa ,
+
and Francesco Melandri*
*Boston Biochem Inc., Cambridge, MA 02139, †UbiQ Bio BV Amsterdam, the Netherlands 1077 XE, +corresponding author
.
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Using Luminescence to Analyze DUB Activity
Ub-KTG Data & Kinetic Parameters
150
1000
+
20
Figure 4: Calculation of UCH-L1 Kinetic Parameters using Ub-KTG FP Substrate.
A: Fluorescence Polarization progress curve for UCH-L1 hydrolysis of Ub-KTG. Ub-KTG
substrate has a 5-tetramethylrhodamine modified Lys-Gly sequence that is linked to Ubiquitin via an
isopeptide bond between the є-amino group of Lysine and Gly76 of Ubiquitin. 100nM UCH-L1 enzyme
was incubated with 100nM Ub-KTG in buffer containing 50mM HEPES pH 7.5, 2mM DTT, 100mM
NaCl, and 0.5mg/mL BSA. 100µL reactions were performed in a black opaque Corning 96-well plate at
25°C. Data was collected on a SpectraMax M5e at 20 second intervals using emission and excitation
wavelengths of 542nm and 580nm, respectively. Fluorescence polarization (mP) values decrease as
the substrate Ub-KTG is converted into KTG product
1000000
Figure 6: Cleavage of Ub-AML by DUBs produces a luminescent signal
Unlike other DUB substrates, Ub-AML is utilized in a coupled reaction to analyze DUB activity.
Aminoluciferin is cleaved from the C-terminus of ubiquitin when in the presence of a DUB. The
liberated aminoluciferin is processed by luciferase and light is generated. The luminescent signal can
be quantitated using a standard plate reader that can process luminescent signals. Using
luminescence as a signal allows for accurate and linear detection of signals over several orders of
magnitude. This allows useful analysis of activity for DUBs that poorly process standard substrates
such as Ub-AMC. However, the coupled nature of the assay that provides such a wide dynamic signal
range prevents using Ub-AML for determining the kinetic parameters of a DUB..
20
0
0
1000
2000
60000
100000
80000
0
100nM UCH-L1
0
100
200
300
400
20000
0
Plane-Polarized Light
(Excitation)
Plane-Polarized Light
(Emission)
KTG
High mP Values
Fast rotation
D: Linear Velocity and Kcat of Ub-KTG by UCH-L1. The spectrum represents only the linear region
of the UCH-L1 progress curve (Figure 4D) and was used to calculate an initial velocity. A linear
regression was performed on these data and the calculated initial velocity = 0.2nM KTG/sec. The
Kcat = 0.002 (0.2nM KTG/sec/100nM UCH-L1).
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Ub-KTG Performance and Comparison
B.
Continuous Fluorescence: 100nM Ub-KTG
versus 100nM Ub-AMC
Figure 1: Fluorescence Polarization (FP) Spectroscopy for Ub-KTG: This figure
depicts the slow rotation of the Ub-KTG FP reagent. The FP reagent is excited by
plane-polarized light and due to its inherent slow rotation the emitted light is largely
plane-polarized. Again, mP signal is a measure of polarized to depolarized light.
Here, the excited and emitted light is highly polarized and therefore produces a large
mP value.
nM (KTG) nM (AMC)
80
60
B
n
40
Enzyme Activity: 0.003 s-1
40nM USP25 with 100nM Ub-AMC
20
Plane-Polarized Light
(Excitation)
KTG
Low mP Values
Figure 2: Fluorescence Polarization (FP) Spectroscopy for KTG Product: This
figure depicts the fast rotation of KTG. When KTG is excited by plane-polarized light
the rotation is much faster by comparison to Ub-KTG. A higher rate of rotation is due
to the much smaller size of KTG versus Ub-KTG. Faster rotation leads to more light
scattering (i.e. emitted light is depolarized). Hence, the mP signal of KTG is lower
than that of Ub-KTG.
KTG
= TAMRA-Lysine-Glysine
KTG
KTG
Isopeptide Bond
DUB
+
+
KTG
e
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io
80
60
40
20
40nM USP25 with 100nM Ub-KTG
0
Fast
Rotation
o
t
s
o
B
Enzyme Activity: 0.004 s-1
0
Scattered Light (Emission)
,
m
Continuous Fluorescence Polarization:
120 USP7FL versus USP7CD
100
nM (KTG)
100
A.
140
500
1000
0
1500
0
Time (seconds)
1000
2000
Enzyme Activity: 2.0E-03 s-1
Enzyme Activity: 3.0E-06 s-1
50nM USP7FL
50nM USP7CD
3000
4000
5000
100
150
200
250
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Figure 5. Performance of Ub-KTG Substrate and Comparing Kinetic Parameters of USP7CD
versus USP7FL. Ubiquitin based FP substrate offers the advantage of a true isopeptide bond between
Ub and fluorophore which currently available Ub-AMC does not have.
A: Performance of Ub-KTG compared to Ub-AMC after calculation of kinetic parameters. 40nM
USP25 enzyme was incubated with 100nM Ub-AMC or Ub-KTG substrate. Both 100μL reactions were
performed in parallel using identical conditions and data processing as described in Figure 4. Based on
these data, comparable enzyme activities were obtained for USP25 with Ub-AMC and Ub-KTG
substrates.
B: Comparison of USP7CD versus USP7FL. 50nM USP7 enzyme or USP7CD were incubated with
100nM Ub-KTG substrate. Both 100μL reactions used identical conditions and data processing as
described in Figure 4 above. Figure 5B clearly demonstrates the increased activity of the full length
enzyme compared to the catalytic core.
Ub-AMC Kcat (sec-1)
Ub-KTG Kcat (sec-1)
FRET (TAMRA/QXL570) (sec-1)
UCH-L1
0.03
0.01
No
UCH-L3
2.7
0.03
No
USP2CD
0.03
3.0E-03
No
USP5
1.58
1.0E-03
1.82
USP7CD
2.8E-03
3.0E-06
No
USP7FL
0.7
2.0E-03
8.7E-03
USP8CD
0.03
6.2E-03
0.02
USP8FL
2.0E-03
1.0E-05
2.5E-04
USP14
No
No
No
USP25
0.003
0.004
No
A20
No
No
No
Ataxin3
No
No
No
BAP1
0.05
2.0E-03
No
Otubain1
No
No
No
1000
10000
USP7 catalytic domain
USP7
USP8 catalytic domain
USP2 catalytic domain
BAP1
1000
0.001
0.01
0.1
1
10
Table 1. Kcat (sec-1) Kinetic Parameters of DUB Enzymes Against Available Substrates.
Conclusions:
 USP7CD has nominal catalytic activity whereas USP7FL has a marked increase in activity.
UCH-L3 enzyme activity>USP5>USP7FL>UCH-L1>USP2CD>USP8CD>BAP1>USP25
Otubain1, A20, Ataxin3, and USP14 have no or minimal activity regardless of substrate
Based on these tabulated data, Ub-AMC, -RH110, -AFC, and Ub-KTG have similar sensitivities,
catalytic rates, as well as differences between core and full length. FP reagents contain a true
isopeptide bond may prove to be a superior alternative to linear fluorescence substrates like UbAMC for future investigation of DUBs.
Figure 9: Ub-AML provides useful signal data over 6 orders of magnitude for signal strength
and DUB concentrations
2-fold serial dilutions of various DUBs were analyzed as described in Figure 7. Reliable observation of
Ub-AML cleavage is observed at submicromolar concentrations even for enzymes that perform very
poorly with Ub-AMC such as USP14 and A20. Of particular note is that significant activity is observed
with the catalytic domain of USP7 which has very poor activity on other DUB substrates as seen in
Figure 5 although it is still an order of magnitude less active than the full length enzyme.
100000
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S/N ratio of luminescence post DUB
processing of UB-AML
10000
0
300
500 pM 250 pM 125 pM 62.5 pM 31.25 pM 15.6 pM 7.81 pM 3.90 pM
pM UCHL3
pM UCH-L3
Figure 7: Linearity of Ub-AML signal over varying concentrations of UCH-L3.
A: Reaction Conditions. All reactions were performed at 37°C using the DUB-Glo™ Protease Kit
from Promega. The peptide substrate provided with the kit was replaced with Ub-AML. Final
concentration of Ub-AML in all reactions was 1.3µM. All DUBs were diluted in a reaction buffer
consisting of 50mM HEPES, pH 7.5, 100mM NaCl, 2mM DTT, and 0.1 mg/ml BSA. 40µl of the
appropriate DUB dilution was placed the into a well of a white, opaque, Costar 96-well half area
plate. The plate was prewarmed at 37°C for 5 minutes before adding 40µl of DUB-Glo reagent with
the appropriate amount of Ub-AML added. Data points represent the mean of 2 samples (n=2). The
80µl reactions were analyzed on a Spectramax M5e for 30 minutes with a reading taken every 45
seconds. The reader was set to detect luminescence at all wavelengths with a 500 msec integration
time.
B: Analyzing linearity of Ub-AML signal for different enzyme concentrations. The luminescence
signals obtained at 360 seconds of a 30 minute run were plotted and fitted by linear regression. The
Ub-AML signal is linear over 3 orders of magnitudes of UCH-L3 concentration.
C: Analyzing linearity of Ub-AML signal over time. RLU signals over a time range of 200-500
seconds during a 30 minute run were plotted for the seven concentrations of UCH-L3 analyzed. The
signal remains stable over time, demonstrating a constant luminescence signal for a defined DUB
concentration when a large excess of substrate is present. The signal is linearly dependent on the
amount of UCH-L3 present.
Time (seconds)
Enzyme
100
10000
40000
6000
USP8CD is much more active as compared to USP8FL.
Figure 3: Hydrolysis of Ub-KTG by a DUB: Overall chemical reaction of Ub-KTG
with a DUB. The Ub-KTG FP reagent has a native isopeptide bond between TAMRA
fluorophore and ubiquitin while other substrates such as AMC are linearly linked to
the C-terminus of Ub.
50
1000
100
USP5
UCHL1
UCH-L3
Ataxin3
A20 catalytic domain
USP14
10
1
0.001
0.01
0.1
1
10
100
1000
10000
nM DUB
100000
S/N ratio of luminescence post DUB
processing of UB-AML
10000
1000
100
USP7 catalytic domain
USP7
USP8 catalytic domain
USP2 catalytic domain
BAP1
10
0.1
1000000
1
10
100
nM DUB
Figure 10: Ub-AML DUB substrate has strong signal to noise ratio.
Data utilized is same as in Figure 8. The average RLU value for each data point was divided by the
average RLU value for reaction buffer and DUB-Glo with 1.3µM Ub-AML. The obtained value is the
signal to noise ratio which was then plotted against the enzyme concentration analyzed. For
comparison, the max signal to noise ratio obtainable in a “standard” Ub-AMC assay with 100nM UbAMC is ~17:1.
100000
RLUs
KTG
o
i
B
y = 194.0x + 985.6
R² = 0.998
o
B
-20000
C: Catalyzed hydrolysis of Ub-KTG by UCH-L1. The mP values for the hydrolysis of Ub-KTG
were converted into nM KTG using the standard curve depicted in Figure 4B, where the production
of one nM KTG represents a decrease of 1.6 mP units.
n
o
t
s
0
500
Time (seconds)
Time (seconds)
Slow Rotation
,
m
e
ch
60000
Slope:0.2 [nM KTG] sec -1
3000
Linearity of Ub-AML signal over varying
concentrations of UCHL3 over time
Linearity of Ub-AML signal over varying
concentrations of UCHL3
40000
20
100nM UCH-L1
100000
nM DUB
40
Enzyme Activity: 2.0E-03 s-1
10000
.
o
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s
o
B
40
Ub-AML Performance and Comparison
RLU
B
n
60
1000
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nc
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60
KTG (nM)
80
KTG (nM)
,
m
100
Ub-AML signal with varying DUBs at different
concentrations
Continuous Fluorescence Polarization: Linear Velocity
80
10
m
100
1
oc
100
Continuous Fluorescence Polarization:
nM KTG Product
0.1
Bi
120
0.01
Light
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D.
10
0.001
nM DUB
B: KTG Product Standard Curve. A standard curve was obtained using a mixture of 100 nM Ub-KTG
and 100nM KTG. The standard curve was prepared in an opaque Corning 96-well plate where each
data point represents the mean of three samples (n = 3). 100µL reactions were read on a SpectraMax
M5e at 542nm excitation and 580nm emission wavelengths. These data range from 100% Ub-KTG
with 0% product to 100% product with 0% Ub-KTG. High mP signals are obtained from samples
comprised of high amounts of Ub-KTG (i.e. 100% Ub-KTG = ~260mP). As product (KTG) is formed
mP signal decreases proportionally (i.e. 100% KTG = ~110mP). A linear regression was performed on
these data and the resulting slope = -1.6 mP per nM KTG produced.
C.
ATP
Mg2+, O2
Luciferase
0
oc
nM (KTG)
60
40
nM (Ub-KTG)
100
Bi
80
80
on
100
60
on
Time (seconds)
40
st
3000
20
,I
100
0
Bo
2000
USP5
UCHL1
UCHL3
Ataxin3
A20 catalytic domain
USP14
m
100
Signal to noise ratio
1000
10000
Signal to Noise Ratio
0
DUBs
Bo
100
100000
he
o
B
150
200
Ub-AML
100nM UCH-L1
st
n
o
t
s
200
mP
100nM UCH-L1
Slope: -1.6 mP/[nM KTG]
nc
250
Ub-AML signal with varying DUBs at different
concentrations
RLU
o
i
B
Enzyme Activity: 2.0E-03 s-1
250
1000000
.
m
e
h
c
Continuous Fluorescence Polarization Standard Curve:
Ub-KTG/KTG Mixture
Continuous Fluorescence Polarization: Raw mP
RLU
300
Fluorescence Polarization Theory
A fluorescent molecule, if stationary and excited by plane-polarized light, will emit
light in the same plane. In reality, fluorescent molecules rotate during the process of
excitation and emission. Smaller fluorescent molecules have a much faster rate of
rotation than large ones. When a small fluorescent molecule is excited by planepolarized light the emitted light is largely depolarized, or scattered. If this same
fluorophore is bound to a large molecule, the overall rotation is markedly slower and
the emitted light is less scattered. Based on these fundamental properties,
Fluorescence Polarization (FP) Spectroscopy is a based on the amount of polarized
and scattered (depolarized) light. The signal is not a measure of relative fluorescent
units (RFU), but millipolarization units (mP). By definition, a mP unit is an average
of polarized light and depolarized light. The experiments described herein utilize a
unique FP reagent based on 5-tetramethylrhodamine (TAMRA) modified Lys-Gly
sequence that is linked to Ubiquitin through an isopeptide bond. This reagent will be
globally referred to as Ub-KTG. Once hydrolyzed by a given DUB the product is LysTAMRA-Gly (KTG).
B.
300
Ub-AML is a substrate for various DUBs
he
A.
mP
Deubiquitinating enzymes (DUBs) play an important role in a multitude of cellular
processes as well as being implicated in a number of human diseases. Despite this
important role, the substrate specificity and kinetic parameters of many DUBs are
poorly defined. This is due to the lack of appropriate reagents where DUB activity can
be monitored in real time while utilizing small amounts of the DUBs themselves which
are often challenging to purify. Ub-AMC has been a workhorse substrate, but the
linkage between fluorophore and Ub doesn’t mimic the isopeptide bond between Ub
and conjugate and many DUBs do not process Ub-AMC rapidly enough to get even
useful qualitative data. Here we describe the development of two new substrates for
analyzing DUB activity that separately address these issues. First, Ub modified by a 5TAMRA-Lys-Gly (Ub-KTG) contains an isopeptide bond between ubiquitin and the
fluorophore, mimicking Ub-conjugation to in vivo substrates. This makes the Ub-KTG
substrate more relevant to understanding how DUBs act during physiological
processes. The extremely photostable fluorophore provides a strong FP signal over
extended time periods. Release of the fluorophore by DUB activity provides a change
in FP signal that can be utilized to determine kinetic parameters. As the FP signal is
concentration independent, low amounts of substrate (100 nM) can be used to obtain
kinetic parameters. Ub-KTG substrate provides similar results to Ub-AMC when tested
against a panel of DUBs. The second substrate developed, Ub-aminoluciferin (UbAML), addresses the issue of sensitivity. Rather than fluorescence as the indicator of
DUB activity, DUB liberated luciferin is processed by luciferase to give a luminescence
signal. Ub-AML not only produces a stronger signal, but also has an excellent signal to
noise ratio over traditional fluorophores. The Ub-AML substrate was used for reliable
detection of DUB activity using an order of magnitude less enzyme than needed for
equivalent results when using Ub-AMC. This makes it possible to rapidly assess the
activity of DUBs that poorly utilize Ub-AMC while using much lower levels of the DUBs
themselves. Both the Ub-KTG and Ub-AML substrates should be useful in providing
new insights into the function of DUBs.
RLU
Introduction
10000
Summary
 Existing Ub-Fluors (Ub-AFC, Ub-AMC, Ub-R110) are still the most trusted mono-Ub fluorescent DUB
substrates available for high-throughput screening (HTS). They provide sensitive and continuous
measurement of enzyme activity for inhibitor screening. However, not all enzymes (DUBs) prefer
these substrates.
1000
100
L1 + Z-RLRGG-AML
L1 + Ub-AML
Figure 8: Ub-AML is processed by UCH-L1 much more effectively than a peptide substrate:
Reactions were performed as described in Figure 7 using 50nM UCH-L1 as the DUB. 40µM of ZRLRGG-AML substrate (provided with the DUB-Glo kit) was used when replacing Ub-AML. Using
Ub-AML as a substrate provides 3 orders of magnitude more signal, demonstrating that Ub-AML is
processed far more quickly by UCH-L1 than peptide substrates. The increased signal is stable over
time (as is the signal resulting from peptide cleavage) even with the faster substrate processing.
This demonstrates that much less enzyme can be used with the Ub-AML substrate while still
obtaining a useful signal.
 Ub-KTG offers the advantage of a highly photostable fluorophore which provides a signal that does
not significantly decay over time. Unlike Ub-AMC, FP Ub-KTG reagent contains an isopeptide bond
which mimics the linkage found between Ub and its substrates in vivo.
 Ub-AML offers the advantage of using a luminescent signal to analyze DUB activity. Coupling the
liberation of aminoluciferin by DUB cleavage with luciferase to process the aminoluciferin and release
light gives a signal with a much wider dynamic range than typical Ub-fluors. Conjugating the
aminoluciferin to Ub rather than a peptide makes the substrate much more amenable to processing
by a wide range of DUBs.
 Ub-AML has much stronger signal strength than Ub-AMC and a higher signal to noise ratio. Ub-AML
also has a 6 order of magnitude range where a linear signal can be detected vs. only 2 for Ub-AMC in
a standard reaction. This is particularly useful for DUBs that have poor activity such as ataxin, USP14,
and A20. Accurate measurement of activity can be obtained using significantly less enzyme than
would be needed in a Ub-AMC assay. Despite the benefits of the stronger signal, Ub-AML cannot be
used to characterize the kinetics of DUBs due to the coupled nature of the system and the rapid
decay of the luminescent signal once it is produced.
 Interestingly, Ub-AML was processed by the USP7 catalytic domain significantly better than it was by
Ub-AMC. This indicates that the Ub-AML may be useful for characterizing the activity of other DUBs
that are not processed effectively by Ub-AMC or other similar substrates.
 These new substrates will help to provide new insight into DUB activity and their physiological roles.