A peptide-based fluorescent probe images ERAAP activity in cells and in high throughput assays

Article abstract of DOI:10.1039/c7cc09598h

ERAAP is an intracellular amino-peptidase that plays a central role in determining the repertoire of peptides displayed by cells by MHC class I molecules, and dysfunctions in ERAAP are linked to a variety of diseases. There is therefore great interest in developing probes that can image ERAAP in cells. In this report we present a fluorescent probe, termed Ep, that can image ERAAP activity in live cells. Ep is composed of a 10 amino acid ERAAP substrate that has a donor quencher pair conjugated to it, composed of BODIPY and dinitro-toluene. Ep undergoes a 20-fold increase in fluorescence after ERAAP cleavage, and was able to image ERAAP activity in cell culture via fluorescence microscopy. In addition, we used Ep to develop a high throughput screen for ERAAP inhibitors, and screened an electrophile library containing 1460 compounds. From this Ep based screen we identified aromatic alkyne-ketone as a lead fragment that can irreversibly inhibit ERAAP activity. We anticipate numerous applications of Ep given its unique ability to image ERAAP within cells.

Full text of DOI:10.1039/c7cc09598h

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A peptide-based fluorescent probe images ERAAP
activity in cells and in high throughput assays†
Cite this:DOI: 10.1039/c7cc09598h
a
Jingtuo Zhang,
Soo Jung Yang,b Federico Gonzalez,b Jiaying Yang,c
Yumiao Zhang,a Maomao He,
Nilabh Shastri *b and Niren Murthy
*
a
a
Received 15th December 2017,
Accepted 31st May 2018
DOI: 10.1039/c7cc09598h
rsc.li/chemcomm
ERAAP is an intracellular amino-peptidase that plays a central role increasing the immune response towards tumors.10,11 Down-
in determining the repertoire of peptides displayed by cells by MHC regulation of ERAAP is also implicated in the development of
class I molecules, and dysfunctions in ERAAP are linked to a variety auto-immune disorders, such as ankylosing spondylitis and
of diseases. There is therefore great interest in developing probes psoriasis.4,5
that can image ERAAP in cells. In this report we present a fluorescent
There is therefore great interest in developing probes that
probe, termed Ep, that can image ERAAP activity in live cells. Ep is can image ERAAP in cells and also in the development of new
composed of a 10 amino acid ERAAP substrate that has a donor inhibitors for ERAAP. However, developing imaging probes for
quencher pair conjugated to it, composed of BODIPY and dinitro- ERAAP has been challenging because it is an intracellular
toluene. Ep undergoes a 20-fold increase in fluorescence after ERAAP amino-peptidase that cleaves large peptides, and developing
cleavage, and was able to image ERAAP activity in cell culture via membrane permeable probes that can turn on fluorescence
fluorescence microscopy. In addition, we used Ep to develop a high after ERAAP mediated cleavage remains a challenge. In addition,
throughput screen for ERAAP inhibitors, and screened an electrophile there are numerous other amino-peptidases within cells that will
library containing 1460 compounds. From this Ep based screen we also potentially cleave fluorescent probes designed to specifically
identified aromatic alkyne-ketone as a lead fragment that can irreversibly detect ERAAP. For example, the only fluorescent probe currently
inhibit ERAAP activity. We anticipate numerous applications of Ep given commercially available for measuring ERAAP activity is leucine-7-
its unique ability to image ERAAP within cells.
amino-5-methylcoumarin (L-Amc), which is cleaved by all amino-
peptidases, and also lacks the optimal emission wavelength for
ER aminopeptidase associated with antigen processing (ERAAP) cellular imaging. Although recent progress has been made in
is an intracellular amino-peptidase, located in the endoplasmic developing ERAP1 (human ortholog of mouse ERAAP) imaging
reticulum, which cleaves peptides presented by the major probes, using two photon emitting dyes,12 there is still a great need
histocompatibility complex (MHC) class I molecules and determines for fluorescent probes that can detect ERAAP via conventional
which subset of peptides is displayed on cell surfaces and fluorescence microscopy.
recognized by killer T cells.1–3 ERAAP thus plays a key role in
In this report we present a fluorescent ERAAP probe,
shaping the immune response and its polymorphic variants composed of a known ERAAP-substrate, the N-terminally
have been implicated in a number of autoimmune diseases.4–6 extended KK-SIINFEKL decapeptide. The modified peptide
Down-regulation of ERAAP results in alterations in the peptides has a donor quencher pair conjugated to it and is termed Ep.
that are displayed on cell surfaces. The altered peptide repertoire is Ep emits at 510 nm after ERAAP cleavage, and can thus image
recognized as foreign, because killer T cells are not tolerant ERAAP activity in cells. In addition, Ep was specific for ERAAP
towards the new peptides.7–9 Therapeutics that can down regulate over other cellular amino-peptidases and has low activity in
ERAAP therefore have great potential as cancer vaccines and cells that lack ERAAP. Finally, Ep has high sensitivity for ERAAP
and was used to develop a high-throughput-screen (HTS) for
a Department of Bioengineering, University of California at Berkeley, Berkeley,
ERAAP inhibitors that allowed us to identify a novel electro-
phile fragment, alkyne-ketone that can inhibit ERAAP.
CA 94720, USA. E-mail: nmurthy@berkeley.edu
b Molecular and Cell Biology, University of California at Berkeley, Berkeley,
CA 94720, USA. E-mail: nshastri@berkeley.edu
The chemical structure of Ep is shown in Fig. 1a, it is
composed of the modified decapeptide K(DNP)-K-S-I-I-N-F-E-
C(BODIPY)-L. Ep contains the fluorescent dye BODIPY and the
fluorescent quencher dinitrophenyl (DNP), conjugated to the
KK-SIINFECL peptide. Ep should initially have low fluorescence
c Chemistry, University of California at Berkeley, Berkeley, CA 94720, USA
† Electronic supplementary information (ESI) available: Detailed procedures for
Ep synthesis, kinetics, quantum yield, docking and Ep-based high throughput
screening. See DOI: 10.1039/c7cc09598h
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Fig. 1 Ep is a peptide-based fluorescent probe that can detect ERAAP. (a) Ep is based on the peptide sequence (KK-SIINFECL) and its fluorescence is
quenched through a FRET pair between DNP and BODIPY. ERAAP cleaves the N-terminal amino acid of Ep and releases the quencher, causing a dramatic
increase in fluorescence. (b) ERAAP activity imaged in wildtype fibroblasts and in ERAAP knockout fibroblasts. (c) A high throughput screening assay
for identifying ERAAP inhibitors developed in a 96-well fluorescent plate reader format. A 1460-electrophile library was screened and we identified para-
bromo-, ortho-fluoro-phenyl ketone alkyne as a lead electrophile fragment that can irreversibly inhibit ERAAP.
because the DNP group will quench the BODIPY fluorescence. and procedures). The final Ep was purified by reverse-phase HPLC
However, in the presence of ERAAP the fluorescence of EP and its synthesis was confirmed by ESI-HRMS (see Fig. S1, ESI†).
should increase, because ERAAP will cleave the N-terminal
Ep is designed to be initially non-fluorescent because of
amino acid modified by the DNP group, due to its amino- DNP mediated quenching, but should increase its fluorescence
peptidase activity. The diffusion of DNP away from the BODIPY after ERAAP cleavage. We therefore investigated the fluorescence
should increase the quantum yield of Ep. The peptide KK-SIINFECL of Ep in its intact state, and its fluorescence after cleavage by Ep.
was selected as the ERAAP substrate for Ep because of its similarity The quantum yield of Ep is extremely low in aqueous solution,
to the well characterized ERAAP substrate KK-SIINFEKL, which is about 1%, due to efficient fluorescence quenching. However,
trimmed by ERAAP to SIINFEKL, the immunodominant H-2Kb- upon addition of ERAAP the fluorescence quantum yield of Ep
binding peptide.3,13 SIINFECL is identical to SIINFEKL except dramatically increases, by a factor of 22 due to ERAAP mediated
that its lysine residue is replaced with a cysteine, which was cleavage, resulting in bright green fluorescence (see Fig. S2 in the
needed for conjugation to BODIPY. Importantly, Serwold et al. ESI†). Fig. 2 demonstrates that the Ep can sensitively detect
demonstrated that the internal lysine in SIINFEKL containing ERAAP and was able to detect ERAAP at concentrations as low as
peptides can be modified to other residues without affecting its 0.1 mg mLꢀ1, and quasi-linearly responded to ERAAP concentrations
ability to bind the Kb MHC I molecule.14,15 Based on this within the sub mg mLꢀ1 range.
design, Ep was synthesized in one step using the commercially
We performed HPLC experiments to determine if Ep was
synthesized peptide K(DNP)K-SIINFECL, which contained the sequentially trimmed by ERAAP at the N terminus to release the
fluorescent quencher, DNP (dinitrophenyl).16–18 The BODIPY DNP residue. Ep was incubated with ERAAP and HPLC was
(4,4-difluoro-3a,4a-diaza-s-indacene), a widely used fluorophore performed in the reaction mixture, after 10 min, and the
for fluorescence imaging in life sciences, was conjugated to this absorption at 350 nm (DNP) and 490 nm (BODIPY) was measured.
peptide via a reaction between the cysteine side-chain and Fig. 3 demonstrates that Ep by itself gives only one peak at 350 nm
iodoacetylamide BODIPY (see the ESI† for the reaction scheme and 490 nm, which overlap. However, after incubation with
ERAAP, the original peak decreased and several new peaks
emerged corresponding to the products of the enzymatic reaction.
We studied the kinetics of the enzymatic trimming reaction
between Ep and ERAAP using fluorogenic assay, by plotting the
initial reaction velocities against Ep concentrations. Ep underwent
Fig. 2 Ep can detect ERAAP activity. The fluorescence intensity of Ep (at
510 nm) changes in response to different concentrations of ERAAP after
incubation in 1ꢁ PBS for 10 min. (Inset: Response curve in the range below
1 mg mLꢀ1 of ERAAP).
Fig. 3 HPLC analysis of Ep before and after trimming by ERAAP.
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typical Michaelis–Menten hydrolysis kinetics (see Fig. S3 in the demonstrated that the N-terminus of Ep has great affinity to the
ESI†), and fitting of the kinetic data to a Lineweaver–Burk plot gave ERAP1 catalytic site, which triggers the trimming process. The
the kinetics parameters Km = 6.7 mM, Vmax = 1.14 nM sꢀ1, and docking result of the C-terminal tripeptide EC(BODIPY)L with
kcat = Vmax/[ERAAP] = 0.13 sꢀ1, corresponding to a catalytic the ERAP1 regulatory site is shown in Fig. 4c. The carboxylate
efficiency (kcat/Km) = 1.94 ꢁ 104 Mꢀ1
s
ꢀ1. The Km and kcat of Ep end of the tripeptide was strongly anchored in the ERAP1
for ERAAP are comparable to other known substrates of ERAAP, regulatory site by direct and water mediated hydrogen bonds
and suggests that it will be able to compete with endogenous with Tyr684, Lys685, Arg807 and Arg841, and the BODIPY residue
cellular substrates for ERAAP in cells.19
on the side chain of the cysteine fell into the non-polar domain
To further understand the binding mechanism of Ep with surrounded by Leu733 and Leu734. This result indicates that the
ERAAP, we performed molecular docking of Ep with ERAAP, C-terminus of Ep can form strong binding with the ERAP1
using the solved crystal structure of ERAP1 (human ortholog of regulatory site. It is also in agreement with the reported binding
mouse ERAAP, PDB 2DY0). ERAP1 has a large and long binding preference of the ERAP1 regulatory site towards hydrophobic
pocket that contains two critical binding sites at its terminal side chains.20
two ends. The Zn-associated catalytic site is at one end of the
There is great interest in being able to image ERAAP activity
binding pocket and binds the N-terminus of the target peptide in cells and we therefore investigated if Ep could allow this. For
and performs the trimming. The other peptide binding site is these experiments we incubated Ep with a fibroblast cell line
the regulatory site (B30 Å away from the catalytic center) which generated from B6 wildtype (WT) mouse and from an ERAAP
binds the C-terminus of the peptide and could function as a knockout (ERAAP-KO) mouse. We treated WT and ERAAP-KO
molecular ruler to control the trimming selectivity. The middle fibroblast cells with 10 or 20 mM of Ep for 2 h or 3 h, and then
part of the binding pocket has a large volume and high imaged via fluorescence microscopy. Fig. 5 demonstrates that
conformational flexibility.19–22
Ep can image ERAAP activity in live cells. For example, strong
We decided to dock only the first 4 and last 3 amino acid fluorescence was detected in WT cells, in contrast, ERAAP-KO
residues of Ep (tetrapeptide and tripeptide) to the active site and cells did not show any observable fluorescence signal, in all Ep
regulatory site, respectively, due to the challenges associated with concentrations and incubation times investigated. These results
docking a large peptide to a flexible pocket in a protein. Fig. 4a demonstrate that Ep can detect ERAAP in cells in a sensitive and
shows the optimized conformation of the tetrapeptide, K(DNP)KSI, specific manner.
in the catalytic site. We found that the N-terminal amine group
The downregulation of ERAP1 (human ortholog of ERAAP)
and the first amide bond were located close to the Zn center and triggers potent anticancer immune responses and can rescue
the carbonyl group was stabilized via hydrogen bonding with mice from experimental models of cancer. Therefore, there is
Tyr438 and His353. The bulky DNP side chain of N-terminal great interest in developing ERAAP inhibitors for cancer
Lys(DNP) fell into the hydrophobic sub-pocket S1 through hydro- immunotherapy.10,11 However, developing ERAAP inhibitors
phobic interactions, the rest of the peptide chain extended back- has been challenging because of the lack of assays available for
ward from the catalytic site. We compared this docking result with detecting ERAAP with sensitivity in a high throughput manner.
the crystal structure of the ERAP1-Bestatin complex (PDB 2DY0). Current ERAAP assays have low selectivity and sensitivity, and are
Bestatin is a widely used Zn metallo-aminopeptidase inhibitor therefore challenging to use for the development of HTS assays for
which tightly binds into the catalytic site and we found that the identifying ERAAP (ERAP1) inhibitors. We therefore investigated if
tetrapeptide K(DNP)KSI and Bestatin shared very similar con- Ep had the sensitivity needed to develop an HTS assay for
formations in the catalytic site (Fig. 4a and b). The results above identifying ERAAP inhibitors. We determined the Z factor of Ep,
with regard to developing ERAAP inhibitors, by measuring its
fluorescence in the presence and in the absence of ERAAP. In a
96-well plate format, this assay gave a Z0-factor of 0.79, which
demonstrates that it is suitable for performing HTS screens
Fig. 4 (a) Optimized confirmations of the tetrapeptide (K(DNP)KSI) in the
ERAP1 catalytic site. (b) Crystal structure of Bestatin in the ERAP1 catalytic
site (PDB: 2DY0). (c) Optimized confirmations of the tripeptide
EC(BODIPY)L in the ERAP1 regulatory site.
Fig. 5 Fluorescence imaging of wildtype (WT) and ERAAP knockout
(ERAAP-KO) fibroblast cells with Ep. WT cells generate green fluorescence
after Ep incubation, whereas ERAAP-KO fibroblasts generate minimal
fluorescence.
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ERAAP activity in cells and in a plate reader format. We
anticipate numerous applications for Ep given the great interest
in understanding the biology of ERAAP and in developing new
ERAAP inhibitors.
Support from NIH grants R37060040 and RO1AI130210 is
acknowledged.
Conflicts of interest
There are no conflicts to declare.
Notes and references
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In conclusion, in this report we present a new peptide based
fluorescent probe for ERAAP, termed Ep, which can image
Chem. Commun.
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