Characterization of Full-Length, Recombinant AMSH/STAM, USP25, and USP9x
Carsten Schwerdtfeger, Ivan Tomasic, Nate Russell, Bradley Brasher, Anthony Mauriello,
Greg Tuffy, Thamara DeSilva and Francesco Melandri
Boston Biochem Inc., Cambridge, MA 02139
AMSH/STAM Hydrolysis of Di-Ubiquitin Chains
MW
+
K6
–
K11
+ –
K27
+ –
K29
+ –
K33
+ –
K48
+ –
K63
+ –
A
linear ;
+ –
A
6kDa
Mono-Ub
Figure 2: AMSH/STAM hydrolysis of various linkages of di-ubiquitin.
2µg of K6, K11, K27, K29, K33, K48, K63, and linear Di-Ub were incubated with 100nM AMSH
and 600nM STAM in 50mM HEPES pH8, 100mM NaCl and 2mM DTT at 37ºC for 60min.
Control reactions contained di-Ub substrate but no AMSH/STAM. Reactions were separated
using 10-20% SDS-PAGE and visualized by Coomassie staining.
AMSH/STAM Hydrolysis of Fluorogenic Ubiquitin
Substrates
Ub-AMC (μM)
Figure 6: USP25 hydrolysis of Ub-AMC and Ub-Rh110.
A: Reactions containing 10nM USP25 were initiated by the addition of Ub-AMC at final
concentrations of 50nM – 8µM. Reactions were carried out in 50mM HEPES pH8, 100mM NaCl
and 2mM DTT at 25ºC. Results are shown in Table 2.
B: USP25 reactions detailed in 6A were repeated, substituting Ub-Rh110 (shown above) or
K63-linked di-Ub FRET (not shown) for Ub-AMC substrate. Results are shown in Table 2.
K11 Di-Ub FRET
Ub-AMC
Figure 10: USP9x hydrolysis of Ub-AMC and Ub-Rh110.
A: Reactions containing 1nM USP9x were initiated by the addition of Ub-AMC at final
concentrations of 50nM – 8µM. Reactions were carried out in 50mM HEPES pH8, 100mM NaCl
and 2mM DTT at 25ºC. Results are shown in Table 3.
B: USP25 reactions detailed in 10A were repeated, substituting Ub-Rh110 (shown above) or
K63-linked di-Ub FRET (not shown) for Ub-AMC substrate. Results are shown in Table 3.
Ub-AMC
Km [µM]
0.7
Kcat [s-1]
0.80
kcat/Km [M-1 s-1]
1.1 x 106
2.4 x 104
Ub-Rh110
1.9
1.14
6.1 x 105
1.4 x 104
K63 Di-Ub FRET
3.8
0.97
2.5 x 105
Ub-AMC
Kcat (s-1)
0.37
kcat/Km (M-1 s-1)
3.9 x 104
Ub-Rh110
12.4
0.30
K63 Di-Ub FRET
7.5
0.11
RFU
USP25 Hydrolysis of Di-Ubiquitin Chains
K6
MW
+
–
K11
+ –
K27
+ –
K29
+ –
K33
+ –
K48
+ –
K63
+ –
USP9x Hydrolysis of Di-Ubiquitin Chains
linear ;
+ –
MW
Domain Structure of AMSH, USP25, and USP9x
Figure 3: AMSH/STAM processing of mono- and di-ubiquitin fluorogenic substrates.
200nM AMSH was pre-incubated with 3µM STAM for 30min at 37ºC. Assays were then initiated
by the addition of 0.5µM Ub-AMC, or 0.5µM K11-, K48-, or K63-Di-Ub FRET substrate. All
reactions were conducted in 50mM HEPES pH8, 100mM NaCl and 2mM DTT at 37ºC.
Ψ
USP
Kinetics of AMSH Activation by STAM
USP9x (2547aa)
SUMO Interacting
Motif
Metalloenzyme
catalytic domain
JAMM/MPN+
Site of poly-sumoylation
in USP25
Ψ
Mono-Ub
6kDa
B
A
B
Ki= 853nM
RFU
SIM
Ubiquitin Interacting
Motif
UIM
Di-Ub
Inhibition of USP9x with Non-Hydrolyzable
DCA-Linked Di-Ubiquitin
Ki= 124nM
Activity at 1µM Inhibitor (%)
Ubiquitin-Like
linear ;
+ –
Inhibition of USP25 with Non-Hydrolyzable
DCA-Linked Di-Ubiquitin
Ubiquitin-Specific
Proteases Domain
USP
K63
+ –
Figure 11: USP9x hydrolysis of various linkages of di-ubiquitin.
2µg of K6, K11, K27, K29, K33, K48, K63, and linear di-Ub were incubated with 1nM USP9x in
50mM HEPES pH8, 100mM NaCl and 2mM DTT at 37ºC for 60min. Control reactions contained
di-Ub substrate but no USP9x. Reactions were separated using 10-20% SDS-PAGE and
visualized by Coomassie staining.
A
V initial (nM s-1)
UBL
Microtubule-Interacting &
Trafficking
MIT
K48
+ –
Figure 7: USP25 hydrolysis of various linkages of di-ubiquitin.
2µg of K6, K11, K27, K29, K33, K48, K63, and linear di-Ub were incubated with 20nM USP25 in
50mM HEPES pH8, 100mM NaCl and 2mM DTT at 37ºC for 60min. Control reactions contained
di-Ub substrate but no USP25. Reactions were separated using 10-20% SDS-PAGE and
visualized by Coomassie staining.
Activation1/2 = 400nM
Ubiquitin Associated
Mono-Ub
6kDa
USP
Domains:
UBA
K33
+ –
Activity at 1µM Inhibitor (%)
UBL
K29
+ –
V initial (RFU s-1)
SIM UIM UIM
UBA
K27
+ –
14kDa
Di-Ub
V initial (RFU s-1)
USP25 (1087aa)
K11
+ –
Reaction Time (seconds)
14kDa
JAMM/MPN+
K6
+ –
21kDa
21kDa
MIT
Ub-Rh110 (μM)
Ub-AMC (μM)
Table 3. Tabulated Steady State Kinetic Parameters of USP9x
Km (µM)
9.4
K48 Di-Ub FRET
USP9x is an essential component of TGFβ/BMP signaling cascade. USP9x biology is likely to
be complex, as increased expression of the DUB correlates with increased MCL1 protein—a
driving force in human follicular lymphoma and diffuse large B-cell lymphomas, whereas
decreased expression of USP9x cooperates with K-ras mutations to accelerate aggressive
pancreatic tumors in mice. This DUB is reported to specifically hydrolyze K29- and K33-linked
polyubiquitins chains, as well as numerous K48-polyubiquitinated substrates.
Ub-Rh110 (μM)
Table 2. Tabulated Steady State Kinetic Parameters of USP25
K63 Di-Ub FRET
V initial (nM s-1)
Di-Ub
V initial (nM s-1)
14kDa
USP25 hydrolyzes ubiquitin-conjugated substrates and may be involved in the processing of
newly synthesized ubiquitin. This DUB is reported to hydrolyze both K48- and K63-linked
polyubiquitin. A muscle-specific USP25 isoform may have a role in the regulation of muscular
differentiation and function. Sumoylation in the vicinity of the tandem UIM domains is reported
to diminish USP25 hydrolysis of polyubiquitin chains.
AMSH (424aa)
B
21kDa
Biology of AMSH, USP25, and USP9x
AMSH is a JAMM-class metalloprotease that specifically cleaves K63-linked polyubiquitin
chains. This DUB is activated by its partner STAM at the endosome, where its activity opposes
ubiquitin-dependent sorting of receptors to lysosomes. AMSH plays important roles in cell
growth, and IL-2, GM-CSF, and BMP (bone morphogenetic protein) signaling pathways.
Kinetic Analysis of USP9x with Fluorogenic
Ubiquitin Substrates
B
V initial (nM s-1)
Attachment of polyubiquitin to substrate proteins generates important biological signaling cues
that are inherent to the linkage type of the polyubiquitin chain. For example, K48-linked
polyubiquitin chains result in proteasome-mediated degradation of proteins to which they are
attached, whereas K63-linked polyubiquitin chains play roles in various intracellular signaling
cascades. An important feature of protein ubiquitination is that it is reversible. Substrateanchored chains may be edited or removed from proteins by specialized proteases called
deubiquitinating enzymes (DUBs). Currently, there are 80-90 DUBs identified in humans and
many have been identified as potential drugable targets because of their involvement in various
disease states. Deubiquitinase activity is often modulated by multiple parameters, including
1) specificity for a protein substrate(s) to which polyubiquitin chains are conjugated, 2) protein
cofactor(s) that may be required for DUB activation, or 3) preference for polyubiquitin linkagetypes.
Thus, understanding the mechanisms, kinetics, and substrate preferences for
deubiquitinases is of great interest, from both academic and clinical viewpoints.
V initial (nM s-1)
Introduction
Kinetic Analysis of USP25 with Fluorogenic
Ubiquitin Substrates
Reaction Time (s)
Substrates for Analyzing DUB Activity
K63-linked DCA Di-Ub (nM)
DCA Di-Ubiquitin Linkage
K63-linked DCA Di-Ub (nM)
DCA Di-Ubiquitin Linkage
STAM1 (μM)
Ub - Leu73 – Arg74 – Gly75 – Gly76
Pro-Fluor
DUB
DUB
Figure 4: Dose Response analysis of STAM stimulating AMSH activity.
200nM AMSH was pre-incubated with 0 - 6.4µM STAM for 30min at 37ºC, then assays were
initiated with the addition of 0.5µM K63 Di-Ub FRET substrate in 50mM HEPES pH8, 100mM
NaCl and 2mM DTT at 37ºC. Half-maximal activation of AMSH occurred at 400nM STAM, and
>90% activation was achieved at 3µM STAM. (Inset: Reaction progress curves for AMSH in the
presence of increasing concentrations of STAM)
Fluor
Ub - Leu73 – Arg74 – Gly75 – Gly76
Lys
Effect of Sumoylation on USP25 Activity
Summary
Ub
B
A
B
A
MW
0
10
20
30
AMSH
USP25
USP9x
K63 only
K11, K33, K48, K63
K6, K11, K29,
K33, K48, K63
No
Yes
Km: 20.9 µM
kcat: 0.7 s-1
kcat/Km: 2.6 x104 M-1s-1
Yes
Km: 0.7 µM
kcat: 0.7 s-1
kcat/Km: 1.1 x106 M-1s-1
Utilizes di-Ub FRET
substrates?
K63 only
Km: 16.5 µM
kcat: 7.7x10-3 s-1
kcat/Km: 4.7x10-1 M-1s-1
K63
Km: 7.5 µM
kcat: 1.1 x10-1 s-1
kcat/Km: 1.4 x104 M-1s-1
K48 not determined
K63
Km: 3.8 µM
kcat: 9.8 x10-1 s-1
kcat/Km: 2.5 x105 M-1s-1
K11, K48 not
determined
Inhibited by DCAlinked di-Ub?
(> 50% inhibition at
1µM DCA di-Ub)
No
Yes
K33, K48, K63, 76-76
Yes
K29, K48, K63, 76-76
40 : Time (min)
DUB
200kDa
USP25-SUMO3n
116kDa
USP25
PIAS2α
Di-Ubiquitin FRET (Isopeptide bond cleavage and release of quenched Fluorophore)
FL
QSY
Ub - Leu73 – Arg74 – Gly75 – Gly76
Lys
QSY
Ub
Lys
Ub
66kDa
55kDa
35kDa
31kDa
FL
Ub-AMC
K63-Di-Ub FRET
USP25 Activity (%)
C
Figure 12: USP9x hydrolysis of Ub-AMC in the presence of non-hydrolyzable di-Ub.
1nM USP9x was pre-incubated for 60min at 37ºC with 0-2µM of K6, K11, K29, K33, K48, K63,
and 76-76 DCA-linked non-hydrolyzable Di-Ub chains. Assays were initiated with the addition of
250nM Ub-AMC substrate in 50mM HEPES pH8, 100mM NaCl and 2mM DTT at 25ºC.
A. Representative dose-response curve for reactions run with K63-linked DCA di-Ub.
B. Percentage of remaining initial activity (vs. uninhibited control reaction) of USP9x in the
presence of non-hydrolyzable DCA di-Ub of various linkages.
.
Ub
Ub
Lys
DUB
Figure 8: USP25 hydrolysis of Ub-AMC in the presence of non-hydrolyzable di-Ub.
10nM USP25 was pre-incubated for 60min at 37ºC with 0-2µM of K6, K11, K29, K33, K48, K63,
and 76-76 DCA-linked non-hydrolyzable Di-Ub chains. Assays were initiated with the addition of
250nM Ub-AMC substrate in 50mM HEPES pH8, 100mM NaCl and 2mM DTT at 25ºC.
A. Representative dose-response curve for reactions run with K63-linked DCA di-Ub.
B. Percentage of remaining initial activity (vs. uninhibited control reaction) of USP25 in the
presence of non-hydrolyzable DCA di-Ub of various linkages.
Kinetic Analysis of AMSH, and AMSH/STAM with
Fluorogenic Di-ubiquitin Substrate
Di-Ubiquitin Chains (Isopeptide bond cleavage)
V initial (nM s-1)
B
Ubiquitin-Fluor’s (C-terminal cleavage releases fluorescent product)
V initial (nM s-1)
A
Di-ubiquitin chain
specificity in
SDS-PAGE assay
Utilizes mono-Ub
fluorogenic substrates
(Ub-AMC)?
21kDa
DUB
D
DUB
Ub
14kDa
6kDa
K63 Di-Ub FRET (µM)
Non-hydrolyzable DCA linked Di-Ubiquitin Chains (Competitive DUB inhibitors )
Ub
Ub - Leu73 – Arg74 – Gly75 – Cys76
DCA
Cys
X
Ub - Leu73 – Arg74 – Gly75 – Gly76
AMC
DUB
Figure 1: Tools for studying deubiquitinating enzymes. Several substrates and substrate
analogs are useful for characterizing deconjugating enzymes in vitro, using kinetic or gel-based
assays. A. Fluorogenic C-terminal derivatives are useful for kinetic studies since the release of
the fluorophore (AMC, AFC, or R110) results in a signal that is directly proportional to activity. B.
Di-ubiquitin substrates are linked via native isopeptide bonds and disassembled in vitro for gelbased end-point assays. C. Fluorophore labeled di-ubiquitin FRET substrates are linked via
isopeptide bonds and disassembled in vitro by DUBS to study chain disassembly in plate-based
real-time assays. D. Non-hydrolyzable dichloroacetone-linked (DCA) di-ubiquitin chains are
competitive inhibitors of some DUBs and may be used to investigate chain specificity of the
enzymes.
SUMO 3
K63 Di-Ub FRET (µM)
Figure 5: AMSH activity assay in the absence or presence of STAM activating protein.
A: 200nM AMSH was pre-incubated for 30min at 37ºC. Assay was then initiated by the
addition of K63-linked Di-Ub FRET substrate at final concentrations of 0.10µM-13µM.
Reactions were carried out in 50mM HEPES pH8, 100mM NaCl and 2mM DTT at 37ºC.
Results are shown in Table 1.
B: 200 nM AMSH plus 3µM STAM were pre-incubated for 30min at 37ºC, then reactions were
conducted as described in 5A. Results are shown in Table 1.
Table 1. Tabulated Steady State Kinetic Parameters of AMSH and AMSH/STAM
AMSH
Km (µM K63 Di-Ub)
16.5
Kcat (s-1)
7.65 x 10-3
kcat/Km (M-1 s-1)
0.47
AMSH + STAM 1
2.4
1.42 x 10-2
5.91
Figure 9. Sumoylation of USP25 and its effect on deubiquitinase activity.
A. Sumoylation reactions containing 400nM SUMO E1, 4µM Ubc9, 500nM PIAS2α, 800nM
USP25, 25uM SUMO3, and 10mM Mg-ATP were incubated at 30°C for indicated times, then
terminated for SDS-PAGE analysis. In subsequent experiments complete sumoylation of
USP25 was achieved (data not shown), and this served as the source of USP25 used in the
deubiquitinase assays described in 9B.
B. Deubiquitinase were set up with 100nM sumoylated or non-sumoylated USP25 using either
250nM Ub-AMC or K63-Di-Ub-FRET substrates at 25°C. Initial velocities were calculated for
each of the four reactions. For each substrate type, the initial velocity of the non-sumoylated
USP25 (control) was defined as 100%, then the velocity of the sumoylated USP25 was plotted
relative to that control. Sumoylated USP25 displayed a 25% reduction in initial velocity
compared to non-sumoylated enzyme in reactions monitored by Ub-AMC. In contrast,
sumoylated USP25 activity against di-ubiquitin was reduced nearly 75% relative to the
unmodified enzyme.
Inhibited by
sumoylation?
Activated by STAM
Yes
N/D
Yes
Km: 2.4 µM
kcat: 1.4 x10-2 s-1
kcat/Km: 5.9 M-1s-1
(kcat/Km ↑ 13-fold)
Vinit with Ub-AMC ↓25%
Vinit with K63 di-Ub FRET ↓75%
N/D
N/D
N/D