Photoaffinity labeling of Ras converting enzyme 1 (Rce1p) using a benzophenone-containing peptide substrate

Article abstract of DOI:10.1016/j.bmc.2010.06.024

Isoprenylation is a post-translational modification that increases protein hydrophobicity and helps target certain proteins to membranes. Ras converting enzyme 1 (Rce1p) is an endoprotease that catalyzes the removal of a three residue fragment from the C-terminus of isoprenylated proteins. To obtain structural information about this membrane protein, photoaffinity labeling agents are being prepared and employed. Here, we describe the synthesis of a benzophenone-containing peptide substrate analogue for Rce1p. Using a continuous spectrofluorometric assay, this peptide was shown to be a substrate for Rce1p. Mass spectrometry was performed to confirm the site of cleavage and structure of the processed probe. Photolysis of the biotinylated compound in the presence of membranes containing Rce1p followed by streptavidin pull-down and Western blot analysis indicated that Rce1p had been labeled by the probe. Photolysis in the presence of both the biotinylated, benzophenone-containing probe and a farnesylated peptide competitor reduced the extent of labeling, suggesting that labeling is occurring in the active site.

Full text of DOI:10.1016/j.bmc.2010.06.024

Bioorganic & Medicinal Chemistry 18 (2010) 5675–5684
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Photoaffinity labeling of Ras converting enzyme 1 (Rce1p) using a
benzophenone-containing peptide substrate
Kelly Kyro a,b, Surya P. Manandhar c, Daniel Mullen a,b, Walter K. Schmidt c, Mark D. Distefano a,b,
*
a Department of Chemistry, University of Minnesota, Minneapolis, MN 55455, United States
b Department of Medicinal Chemistry, University of Minnesota, Minneapolis, MN 55455, United States
c Department of Biochemistry and Molecular Biology, University Georgia, Athens, GA 30602, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 21 January 2010
Revised 4 June 2010
Accepted 7 June 2010
Available online 12 June 2010
Isoprenylation is a post-translational modification that increases protein hydrophobicity and helps target
certain proteins to membranes. Ras converting enzyme 1 (Rce1p) is an endoprotease that catalyzes the
removal of a three residue fragment from the C-terminus of isoprenylated proteins. To obtain structural
information about this membrane protein, photoaffinity labeling agents are being prepared and
employed. Here, we describe the synthesis of a benzophenone-containing peptide substrate analogue
for Rce1p. Using a continuous spectrofluorometric assay, this peptide was shown to be a substrate for
Rce1p. Mass spectrometry was performed to confirm the site of cleavage and structure of the processed
probe. Photolysis of the biotinylated compound in the presence of membranes containing Rce1p followed
by streptavidin pull-down and Western blot analysis indicated that Rce1p had been labeled by the probe.
Photolysis in the presence of both the biotinylated, benzophenone-containing probe and a farnesylated
peptide competitor reduced the extent of labeling, suggesting that labeling is occurring in the active site.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Benzophenone
Rce1p
Prenyl protease
Proteolysis
Protein isoprenylation
Photoaffinity labeling
CaaX processing
1. Introduction
Ca1a2X sequence by either protein farnesyltransferase (PFTase) or
protein geranylgeranyl transferase (PGGTase). The -X residue of
the Ca1a2X sequence influences the identity of the isoprenoid,
and -a1a2 are typically aliphatic amino acids.2,3,5 Ca1a2X-containing
1.1. Sequential processing of ras proteins
Protein isoprenylation, proteolysis, and carboxylmethylation
are sequential post-translational modifications that occur to ras
proteins. These modifications increase protein hydrophobicity, as-
sist in membrane localization, and facilitate signal transduction.1–4
Ras is a GTP-binding protein (G-protein) that contains a Ca1a2X
motif. The post-translational processing of Ca1a2X proteins
(Fig. 1) first involves isoprenylation at the cysteine residue of the
isoprenylated proteins are then modified at the C-terminus by the
Ca1a2X Box
O
R1
O
R3
H
N
H
N
OH
Protein
N
H
N
H
O
R2
O
S
Abbreviations: Abz, 2-aminobenzoyl; C5BP, 3-methylbenzophenone prenyl
ether; CLEAR, crosslinked ethoxylate acrylate resin; Dde, 1-(4,4-dimethyl-2,6-
dioxocyclohexylidene) ethyl; DI, deionized; DIEA, diisopropylethylamine; DMF,
N,N-dimethylformamide; DMSO, dimethyl sulfoxide; Dnp, dinitrophenyl; ECL,
enhanced chemiluminescence; ER, endoplasmic reticulum; ESI-MS, electrospray
ionization mass spectrometry; FA, formic acid; Fmoc, 9-fluorenylmethyloxycar-
bonyl; Fr, farnesyl; HA, hemagglutinin; HRP, horseradish peroxidase; BME, 2-
mercaptoethanol; OAc, acetate; PBS, phosphate buffered saline; PEG, polyethylene
glycol; PVDF, polyvinylidene difluoride; PMSF, phenylmethanesulfonyl fluoride;
RIPA, radioimmunoprecipitation assay; RP-HPLC, reversed-phase high pressure
liquid chromatography; SA, streptavidin; SPPS, solid-phase peptide synthesis; TBST,
Tris-buffered saline containing Tween-20; TFA, trifluoroacetic acid; TFP,
tetrafluorophenyl.
Site of Proteolysis
Ras Converting Enzyme (Rce1p)
O
H
N
Protein
OH
S
Figure 1. Rce1p proteolysis of the last three residues of a C-terminal tetrapeptide
Ca1a2X motif. C, cysteine; R1R2, any aliphatic amino acid; R3, one of several amino
acids.
* Corresponding author. Tel.: +1 612 624 0544, fax: +1 612 626 7541.
E-mail address: diste001@umn.edu (M.D. Distefano).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.06.024

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K. Kyro et al. / Bioorg. Med. Chem. 18 (2010) 5675–5684
protease, Ras converting enzyme 1 (Rce1p), which removes the -
a1a2X tripeptide in the endoproteolysis step (Fig. 1). This is fol-
lowed by carboxylmethylation of the C-terminal prenyl cysteine
by the isoprenylcysteine carboxymethyltransferase (Icmt), also
called Ste14p in yeast.1,2,4–8
mutagenesis revealed that three conserved residues (Glu156
,
His194, and His248) were required for Rce1p activity.1,10 The confir-
mation that Rce1p was inhibited by 1,10-phenanthroline and ex-
cess zinc offered additional support for the metal-dependent
protease mechanism.8,10 The evidence that Rce1p proteolysis re-
quires histidine and glutamate residues does not, however, rule
out the possibility that Rce1p utilizes a yet undiscovered protease
mechanism.
The mechanism of catalysis for Rce1p remains undefined, partly
because its membrane association makes it difficult to purify.
Rce1p localizes at the ER and is predicted to have multiple mem-
brane spanning regions.1,8,9 The critical catalytic residues of Rce1p
are purported to be embedded in the membrane to facilitate inter-
action with isoprenoid-modified substrates, but the structure and
topology of Rce1p have yet to be resolved.10 Rce1p is involved in
the membrane localization of all known forms of human ras and
inhibition of its protease activity alters ras function.5 Mutated
forms of ras proteins are associated with cancerous signal trans-
duction in roughly 15–20% of human cancers. Consequently, dis-
ruption of Ca1a2X proteolysis is viewed as an important
therapeutic strategy in targeting ras-mediated cancers.1,4
Mutation, inhibition, and bioinformatic studies have been per-
formed to determine the mechanism and protease class of Rce1p.
Independent studies suggest that Rce1p is either a metalloprotease
or a cysteine protease, but it is still possible that Rce1p belongs to a
yet undiscovered class of proteases.1,8,10–12 The cysteine protease
classification for Rce1p has been questioned by mutational and
bioinformatic investigation.8,10,12 Even though Rce1p is reported to
require a conserved cysteine (Cys251) for activity,1 experimental
analyses maintain that Cys251 is not required for Rce1p func-
tion.8,10,12
To begin to address the issue that currently, no structural infor-
mation exists for Rce1p,10 we have designed substrate analogues
that incorporate photoactive isoprenoids that can potentially be
used to label and identify active site residues.
1.2. Design of Rce1p substrate analogue incorporating a
benzophenone photophore into the prenyl group
Isoprenylated peptide substrate, 2 ( Fig. 2), derived from the
C-terminal sequence (CVIM) of mammalian K-Ras4B, has been an
effective substrate in Rce1p proteolysis assays.13 This farnesylated,
nona-peptide contains an N-terminal Abz (2-aminobenzoyl) fluo-
rophore and a dinitrophenyl quencher (lysine e-dinitrophenyl) is
attached at the a1 (V) position.8 It has been previously reported
that both KSKTKC(Fr)VIM and Abz-KSKTKC(Fr)VIM are equally
good substrates for Rce1p, exhibiting identical kinetic parameters
(KM = 4 lM; Vmax = 600 nmol/h/mg Rce1p-containing membrane
protein) and indicating that the fluorophore has no effect on pep-
tide proteolysis.9,10,13 Vmax decreased by half (Vmax = 300 nmol/h/
mg Rce1p-containing membrane protein) when the quencher
was linked at the V position, but the peptide was still an efficient
substrate.13
Sequential analysis of Rce1p identified invariant residues remi-
niscent of a consensus HEXXH motif found in metalloproteas-
es.1,10,12 Metal-dependent enzymes require two histidine residues
and at least one glutamate residue for activity.10 Site-directed
Compound 4 (Fig. 3) is an analogue of 2, containing a photoac-
tive benzophenone-modified isoprenoid linked to the peptide. The
NO2
NH2
NH2
NH2
HN
NO2
S
O
O
O
O
S
O
NH2
H
N
H
N
H
N
H
N
OH
N
H
N
H
N
H
N
H
N
H
O
O
O
O
O
OH
OH
2
Figure 2. Structure of isoprenylated peptide substrate, 2, derived from the C-terminal sequence (CVIM) of mammalian K-Ras4B.
NO2
NH2
NH2
NH2
HN
NO2
S
O
O
O
O
S
O
NH2
H
N
H
N
H
N
H
N
OH
N
H
N
H
N
H
N
H
N
H
O
O
O
O
O
O
OH
OH
4
O
Figure 3. Structure of substrate analogue, 4, incorporating a benzophenone-modified isoprenoid.

K. Kyro et al. / Bioorg. Med. Chem. 18 (2010) 5675–5684
5677
benzophenone group is tethered to the isoprenoidal portion of the
probe by a stable ether bond to permit photocrosslinking of
Rce1p.7,14,15
(m/z = 676.3) (Fig. 5A). For peptide 4, similar results were observed.
Fragmentation of the [M+3H]3+ species resulted in the production
of a number of a-, b-, and y-type ions; four out of nine b-type ions
and six out of nine y-type ions were observed (see Table 3 in the
Supplementary data), all consistent with the desired peptide 4.
As was noted with 2, a large number of ions where loss of the ben-
zophenone-isoprenoid moiety had occurred were observed; those
include ions related to the doubly and triply charged parent ions
(m/z = 709.5 and m/z = 473.3) (Fig. 5B). A very intense ion from
the benzophenone fragment (BP) (m/z = 195.1) was also observed.
This fragmentation may be useful in identifying specific cross-
linked peptides in future MS–MS analysis.
For photolabeling applications and subsequent pull-down
experiments, the biotinylated peptide 6 (Fig. 6) was synthesized.
This was accomplished by first preparing 5 (Fig. 6), which contains
a biotin group linked to the peptide N-terminus via a PEG spacer.
Peptide 5 also contains a orthogonally-protected lysine (using a
Dde group) to allow for the introduction of a fluorophore in future
experiments. Peptide 6 was obtained by alkylation of 5 with 3
(Fig. 4) using conditions analogous to those noted above.
Benzophenone photophores have proven efficacy as monoden-
tate photoaffinity labeling tools.16 They combine stability with
specificity and allow enough photoincorporation for product
identification.17 The aryl ketone probes can be crosslinked to mem-
branes in order to map their hydrophobic surface.7 The isoprenoid-
linked aryl ketone photophore ( Fig. 3) bears enough structural
similarity to a C15 isoprenoid to possibly be useful for protein
incorporation.18,19 While other types of photoactive isoprenoids
have been prepared including diazo esters20–25 and aryl azides,26
we chose to employ the benzophenone-based structures due to
their ease of synthesis and their ability to be excited at wave-
lengths above 300 nm. It should be noted that while a plethora
of modified isoprenoid diphosphates have been used to study
prenyltransferases and interactions between prenylated proteins
and other proteins,27,28 the use of photoactive isoprenoid ana-
logues to probe the processing enzyme, Rce1p, remains a largely
unexplored approach. Here we describe the utility of one such mol-
ecule and show that it is an alternative substrate for Rce1p. Irradi-
ation of this probe in the presence of partially-purified membranes
containing Rce1p results in selective labeling of the target enzyme.
2.2. Fluorescence-based proteolysis assay for kinetic analysis of
farnesylated and benzophenone-modified probes
2. Results and discussion
A previously-described, fluorescence-based proteolysis assay
has been used to evaluate proteolytic processing of prenylated
peptide substrate 2.9,13 Cleavage of the C-terminal -K(Dnp)IM tri-
peptide results in a time-dependent increase in fluorescence of
the Abz fluorophore (420 nm) that is used to monitor the rate of
endoproteolysis. This fluorogenic assay was used to evaluate 4 as
an alternative substrate for Rce1p. For comparative purposes, as-
says were also performed with 2.1,8,9,13
Carbonate-washed membranes enriched for Rce1p were iso-
lated from yeast over-expressing Rce1p as the sole Ca1a2X prote-
ase; the structurally unrelated Ste24p/Afc1p protease cleaves
certain Ca1a2X sequences, but not those in Ras-based substrates.5
The Rce1p-enriched membranes were added to a solution contain-
ing the peptide substrates; a fluorescence plate reader was used to
monitor Rce1p activity. Kinetic evaluation demonstrated that 4 is a
2.1. Synthesis of Rce1p substrates
Peptide 1 (Fig. 4) was synthesized by Fmoc-based solid-phase
peptide synthesis prior to alkylation with farnesyl bromide, using
Zn2+ catalysis under acidic conditions to create peptide
2
(Fig. 2).29 probe 4 (Fig. 3) was prepared in a similar fashion using
bromide 3 (Fig. 4).18,20
Peptides were purified by RP-HPLC and characterized by mass
spectrometry. ESI-MS–MS of spectra of 2 and 4 are shown in Figure
5. For peptide 2, the most abundant [M+3H]3+ species (m/z = 519.4)
was fragmented to yield a large number of a-, b-, and y-type frag-
ments; four out of nine a-type ions, six out of nine b-type ions, and
six out of nine y-type ions were observed, all consistent with the
proposed structure of 2 (see Table 2 in the Supplementary data
for complete listing and assignments). Interestingly, a large num-
ber of fragments where loss of the farnesyl group had occurred
were observed. The most abundant ion in the ESI-MS–MS was
the doubly-charged parent ion that had lost the farnesyl group
Rce1p substrate with a KM of 1.9 lM and a Vmax of 0.093 nmol/min/
mg protein (Table 1, Fig. 7B). Comparison of those values with anal-
ogous kinetic parameters obtained with 2 (Table 1, Fig. 7A) sug-
gests that 4 binds to Rce1p with higher affinity (3.4-fold) than 2
but that the maximal rate is approximately 60-fold lower. For
NO2
NH2
NH2
NH2
HN
NO2
S
NH2
O
O
O
O
O
H
N
H
N
H
N
H
N
OH
N
H
N
H
N
H
N
H
N
H
O
O
O
O
O
OH
OH
SH
1
O
Br
O
3
Figure 4. Structures of precursor compounds 1 and 3. Peptide 1, containing a free cysteine thiol, was alkylated with farnesyl bromide to produce peptide 2. The
benzophenone-modified isoprenoid, 3, was used for alkylation of 1 to produce the aryl ketone-containing peptide photophore, 4.

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K. Kyro et al. / Bioorg. Med. Chem. 18 (2010) 5675–5684
A
Compound 2
350
300
250
200
150
100
50
[M+2H]2+(-f)
b2
b1
y1
y8(-f)
2+
y9
2+
a3
y72+(-h)
a7(-i)
b2(-h)
y2
a92+(-f)
y82+(-f)
y9(-f)
b9(-f)
[M+2H]2+(-g)
a9
2+
y6(-f)
900
[M+H]+(-f)
b3
b82+(-f)
y7(-f)
1100
b4
500
0
100
300
700
m/z (amu)
1300
Compound 4
B
b1
BP
140
120
100
80
y1
[M+3H]3+
[M+2H]2+(-b)
b2
60
2+
2+
2+
y7
y8
y9
40
[M+2H]2+(-d)
2+
[M+3H]3+(-d)
500
y
b2(-h)
y2
b92+(-d)
6
2+
20
b3
2+
b9
y8(-b)
b3
0
100
300
700
m/z (amu)
900
1100
1300
Figure 5. ESI-MS–MS of Rce1p substrates. (A) Farnesylated substrate 2 (-f, loss of farnesyl (C15H25); -g, loss of farnesyl-H2O (C15H27O); -h, loss of H2O; -i, loss of NH3).
(B) Benzophenone-modified substrate
(BP, benzophenone fragment, (3-benzoylphenyl)methylium (C14H11O+); -b, loss of benzophenone fragment; -d, loss of
benzophenone–H2O, (3-(hydroxymethyl)phenyl)(phenyl)methanone (C14H13O2); -h, loss of H2O).
4
several reasons, the value for Vmax for 2 differs from that previously
reported.8,30 First, there is the impact of curve fitting to the Hill
equation instead of the Michaelis–Menten equation as done previ-
ously; the Hill equation provides a better fit for these and past data.
Second, Vmax is reported per mg of membrane protein, whereas
previous values were not adjusted in this manner. Identical analy-
sis of previous data (i.e., Hill equation and normalization to mg of
protein) yields Vmax values approximately half that reported here
(2.5 and 2.8 vs 5.6 nmol/min/mg). We attribute the higher Vmax
value in this study to the use of membranes better enriched for
Rce1p activity (i.e., carbonate-washed membranes).
were obtained with the product derived from Rce1p-catalyzed pro-
teolysis of 4. The dominant [M+2H]2+ species (m/z = 546.4) in the
ESI-MS is consistent with the expected product 8 (Fig. 8). MS–MS
fragmentation of that ion ( Fig. 9B) produced a full set of both
b- and y-type ions all consistent with the proposed structure for
8. As was noted for 7, cleavage of the thioether bond linking the
isoprenoid moiety to the cysteinyl thiol constitutes a major mani-
fold of fragmentation in these compounds.
2.4. Photoaffinity labeling of Rce1p using biotinylated,
photoactive peptide substrate 6
2.3. Mass spectrometric analysis of enzymatic processing of
farnesylated and benzophenone-modified probes
With the confirmation that Rce1p can act on a substrate con-
taining a photoactive isoprenoid, the related peptide 6 that incor-
porates a biotin moiety was employed in photoaffinity labeling
experiments. A carbonate-washed membrane fraction fromSaccha-
romyces cerevisiae was used as the source for Rce1p (RCE1). A sim-
To confirm the identity of the product obtained from Rce1p-cat-
alyzed proteolysis of 4, a large-scale (500 lL) incubation of 4 with
Rce1p was performed. Soluble products were purified using re-
versed phase chromatography and analyzed by ESI-MS. A similar
large-scale reaction and analysis was carried out with 2 to compare
the MS fragmentation. Direct infusion of the products obtained
from Rce1p-catalyzed proteolysis of 2 revealed that the most abun-
dant [M+2H]2+ species (m/z = 509.4) in the ESI-MS was consistent
with the expected product 7 (Fig. 8). Fragmentation of that ion
via MS–MS (Fig. 9A) resulted in the formation of a-, b-, and y-type
ions, as was observed with 2; in total, one out of six a-type ions,
four out of six b-type ions, and one out of six y-type ions, were ob-
served. Cleavage of the thioether bond linking the farnesyl group to
the peptide was a common mode of fragmentation. Similar results
ilar membrane fraction isolated from a strain lacking Rce1p (rce1D)
was employed for negative control experiments; the strains also
lack Ste24p in both cases. Photolysis reactions were conducted at
4 °C using 6 at saturating concentrations.19,20,31 The membrane
protein fractions were irradiated, concentrated by ultracentrifuga-
tion, and treated with streptavidin beads to capture any cross-
linked proteins. After elution, the resulting proteins were
fractionated by SDS–PAGE and analyzed by Western blotting using
antibodies directed against HA, an epitope tag incorporated into
Rce1p via recombinant methods.10
Irradiation of 6 in the presence of membranes containing Rce1p
resulted in the appearance of an intense band at approximately

K. Kyro et al. / Bioorg. Med. Chem. 18 (2010) 5675–5684
5679
NO2
NH2
NH2
NH2
HN
NO2
S
O
O
O
O
O
O
H
N
H
N
H
N
H
N
H
N
OH
N
H
N
H
N
H
N
H
N
H
N
H
Biotin-(PEG)3
O
O
O
O
O
O
OH
OH
SH
5
HN
O
O
NO2
NH2
NH2
NH2
HN
NO2
S
O
O
O
O
O
O
H
N
H
N
H
N
H
N
H
N
OH
N
H
N
H
N
N
N
N
H
Biotin-(PEG)3
H
H
H
O
O
O
O
O
O
O
OH
OH
6
S
O
HN
O
O
Figure 6. Structures of biotinylated peptides 5, containing a free thiol, and 6 following alkylation using the benzophenone-modified isoprenoid, 3.
Table 1
40 kDa (Fig. 10, Lane 3), consistent with the predicted mass of HA-
epitope tagged Rce1p (40.75 kDa). This data indicates that Rce1p
can be effectively captured upon crosslinking to a biotinylated
probe. Importantly, no HA-containing bands were detected in the
photolysis reaction performed on membranes lacking Rce1p
(Fig. 10, Lane 4). A small amount of Rce1p was observed in the ab-
sence of irradiation (Fig. 10, Lane 1) that was not present in the
membranes lacking Rce1p (Fig. 10, Lane 2), indicating that the en-
zyme has some nonspecific affinity for the streptavidin beads used
Kinetic parameters for substrate activity of Rce1p with benzophenone-modified
photoprobe 4 and farnesylated peptide 2.
Substrate
Kinetic parameters
Vmax (nmol/min/mg
protein)
Vmax/KM (min/mg
protein)
RM
M)
(
l
2 (n = 2)
4 (n = 5)
6.4 0.2
5.6 0.1
5.1 ꢀ 10ꢁ4
1.9 0.3 0.093 0.005
4.8 ꢀ 10ꢁ5
A
B
8
0.15
0.1
0.05
0
6
4
2
0
0
12.5
25
[2] (µM)
37.5
50
0
12.5
25
37.5
50
[4] (µM)
Figure 7. Kinetic analysis of Rce1p-catalyzed hydrolysis of farnesylated peptide, 2, and benzophenone-modified substrate 4. (A) Rate versus substrate concentration using
peptide 2. (B) Rate versus substrate concentration using peptide 4.

5680
K. Kyro et al. / Bioorg. Med. Chem. 18 (2010) 5675–5684
NH2
NH2
NH2
NH2
NH2
NH2
O
O
O
O
S
O
O
O
O
S
NH2
NH2
H
N
H
N
H
N
H
H
N
H
N
N
N
N
N
OH
N
N
N
OH
H
H
H
H
H
H
O
O
O
O
O
O
O
OH
OH
OH
OH
7
8
O
Figure 8. Structures of products 7 and 8 derived from Rce1p-catalyzed proteolysis of 2 and 4, respectively.
Compound 7
A
[M+H]+(-f)
50
40
30
20
10
0
b1
b2
[M+2H]2+(-f)
[M+2H]2+
b6
b2(-h)
250
y5(-f)
b4
2+
y5
a3
100
400
550
m/z (amu)
700
850
1000
Compound 8
B16
[M+2H]2+(-c)
[M+2H]2+
b2
b1
BP
14
12
10
8
y5
6
[M+2H]2+(-d)
b4(-h) a5
b2(-h)
4
b5
y1
2+
y5(-d) b6
700
2+
b3(-h)
2
b6
b3
y5
[M+H]+(-b) y6
900
y2
y4
y3
0
100
300
500
m/z (amu)
Figure 9. ESI-MS–MS of products obtained from Rce1p-catalyzed proteolysis of substrate peptides 2 and 4. (A) Farnesylated proteolysis product 7 (-f, loss of farnesyl (C15H25); -h,
loss of H2O). (B) Benzophenone-modified proteolysis product 8 (BP, benzophenone fragment, (3-benzoylphenyl)methylium (C14H11O+); -b, loss of benzophenone fragment; -c,
loss of (E)-(3-(((2-methylbut-2-en-1-yl)oxy)methyl)phenyl)(phenyl)methanone (C19H20O2); -d, loss of benzophenone–H2O, (3-(hydroxymethyl)phenyl)(phenyl)methanone
(C14H13O2); -h, loss of H2O).
in the pull-down process. That nonspecific binding was decreased
by raising the detergent concentration in the wash step of the pull-
down experiment from 0.1% SDS (data not shown) to 1.5% SDS (see
Fig. 10) but could not be completely eliminated. It is unlikely, how-
ever, that the weak nature of nonspecific binding will interfere in
subsequent efforts to identify the site of crosslinking.
was reduced to 0.75
used. Thus, irradiation of 6 in the presence of 50
in a significant reduction (60% of that observed in the absence of
2) in crosslinked Rce1p (Fig. 11, Column 2). The extent of crosslink-
ing by 6 was further reduced (to 31% of that observed in the ab-
l
M. This allowed a larger excess of 2 to be
M 2 resulted
l
sence of 2) by increasing the concentration of 2 to 100 lM in the
photolysis reaction (Fig. 11, Column 3). Overall, these results are
consistent with the conclusion that probe 6 is labeling the active
site of Rce1p.
2.5. Competition experiments using 2 and 6
Next, to examine the specificity of 6, we studied the ability of
the benzophenone-containing probe to label Rce1p upon photoly-
sis in the presence of a competitor. Farnesylated peptide 2, a
known substrate for Rce1p, was selected as the competitor. Due
to the limited solubility of 2, the concentration of 6 employed
3. Conclusions
Rce1p catalyzes the proteolytic removal of C-terminal tripep-
tides from prenylated proteins and is a possible target for the

K. Kyro et al. / Bioorg. Med. Chem. 18 (2010) 5675–5684
5681
for the related farnesylated peptide (2). However, with a KM of
1.9 M, 4 demonstrated 3.4-fold higher binding affinity than 2.
kDa
220
1
2
3
4
l
ESI-MS–MS was employed to confirm the site of proteolysis. It
should be noted that the inclusion of the Abz fluorophore and
Dnp quencher in 4 greatly simplified kinetic analysis and purifica-
tion of the cleaved product for subsequent structural analysis. Pho-
tolysis of a biotinylated version of the probe (6) followed by
streptavidin pull-down, SDS–PAGE and Western blot analysis dem-
onstrated that Rce1p can be labeled by the probe and that the
resulting crosslinked protein can be isolated. Photolysis in the
presence of both probe 6 and a farnesylated peptide competitor
2 reduced the extent of labeling. Taken together, these results
demonstrate that benzophenone-containing peptides can serve as
bone fide substrates for Rce1p and suggest that the enzyme is
being labeled in the active site. Efforts to use this probe to identify
residues in the active site of Rce1p are ongoing.
100
60
45
30
20
12
8
Figure 10. Western blot analysis of photolabeling of Rce1p with probe 6 detected
with anti-HA following SA pull-down and SDS–PAGE separation. Lane 1: Rce1p-
4. Experimental
4.1. General
containing membranes (RCE1) and 6 (15
lacking Rce1p (rce1 ) and 6 (15 M), no UV irradiation. Lane 3: Rce1p-containing
membranes (RCE1) and 6 (15 M), with UV irradiation. Lane 4: membranes lacking
Rce1p (rce1 ) and 6 (15 M), with UV irradiation. Results shown are representative
lM), no UV irradiation. Lane 2: membranes
D
l
l
D
l
of those obtained in three independent experiments.
Analytical TLC was performed on precoated (0.25 mm) Silica Gel
60F-254 plates purchased from E. Merck. Flash chromatography
silica gel (60–120 mesh) was obtained from E. M. Science.
Fmoc-Cys(Me)-OH was from Bachem and Fmoc-Lys(Abz)-OH,
Fmoc-Lys(Dnp)-OH, Fmoc-Lys(Dde)-OH were from Nova Biochem.
Preloaded CLEAR-Acid resins were from Peptides International.
TFP–PEG3–biotin was from Thermo Scientific. All other reagents
were from Sigma–Aldrich. Reactions were performed at room tem-
perature unless otherwise noted. RP-HPLC analysis was carried out
using a Beckman Model 125/168 instrument equipped with a
diode array UV detector. Preparative RP-HPLC separations em-
120
2
(0 µM)
100
80
2
(50 µM)
60
2
ployed a Phenomenex Luna 10
mm ꢀ 25 cm). Analytical RP-HPLC separations employed a Varian
Microsorb-MV 5
100 Å C18 column (4.6 mm ꢀ 25 cm) with a
Phenomenex Luna 5
l 100 Å C18(2) column (10.00
40
20
0
(100 µM)
l
l
guard cartridge (4.6 mm ꢀ 3 cm). Retention
times (tR) were based on analytical RP-HPLC using a linear gradient
from 100% H2O to 40% H2O/60% CH3CN over 60 min and a flow rate
of 1.0 mL/min. Peptide concentrations were determined by UV
1
2
3
spectroscopy of the Dnp quencher (349 nm,
e
= 18,000 cmꢁ1 Mꢁ1).
Figure 11. Densitometric quantification of western blot analysis of photolabeling of
Rce1p with probe 6 in the presence of competitor 2 detected with anti-HA following
SA pull-down and SDS–PAGE separation. Column 1: Rce1p-containing membranes
The fluorogenic peptides were protected from light as much as
possible to avoid bleaching of the Abz fluorophore. C18 Sep-Pak col-
umns were purchased from Waters. Protein precipitation kit was
purchased from CalBiochem. Spin columns, streptavidin resin,
and ECL substrate solutions were purchased from Pierce. Autora-
diographic film was purchased from Bioexpress. Antibodies were
purchased from Covance and GE Healthcare.
(RCE1) and probe 6 (0.75
membranes (RCE1), probe 6 (0.75
UV irradiation. Column 3: Rce1p-containing membranes (RCE1), probe 6 (0.75
and farnesylated competitor 2 (100 M) with UV irradiation.
l
M) with UV irradiation. Column 2: Rce1p-containing
M), and farnesylated competitor 2 (50 M) with
M),
l
l
l
l
design of anti-cancer agents. Site-directed mutagenesis has been
used to identify conserved residues that may be required for Rce1p
catalytic activity.1,10 Both competitive inhibitors and active site di-
rected, irreversible affinity labeling reagents have been designed to
characterize the structure and identify the mechanistic class of
Rce1p.11,32,33 Photoaffinity labeling reagents represent an addi-
tional means to evaluate this membrane-associated protease. The
introduction of a photolabile moiety to the prenylcysteine of sub-
strate peptides offers potential utility for structural analysis of
Rce1p. The benzophenone-containing compounds are reasonable
structural mimics of their farnesylated counterparts and their abil-
ity to crosslink to neighboring residues upon photoactivation
makes them an appealing tool.
To gain further understanding into the structure of Rce1p, a
peptide substrate containing a benzophenone-functionalized iso-
prenoid was designed, prepared, and tested to study its efficacy
as a Rce1p substrate. Using a continuous spectrofluorometric assay
it was shown that this substrate (4) is processed by Rce1p with an
efficiency (Vmax/KM) that is approximately 18-fold lower than that
4.1.1. Yeast strains and plasmids
The parent yeast strain used in this study is SM3614 (MATa trp1
leu2 ura3 his4 ste24::LEU2rce1::TRP1).34 SM3614 was transformed
with plasmid pRS316 (CEN URA3) or pWS479 (2
lURA3PPGK-RCE1::-
HA) to create rce1
D
and wildtype (WT) strains, respectively.10,35
Yeast transformations were carried out according to established
methods.36 Transformed yeast strains were grown at 30 °C in syn-
thetic complete medium lacking uracil (SC-Ura).
4.2. Chemical synthesis
4.2.1. Synthesis of Abz-KSKTKCK(Dnp)IM, 1
The linear peptide sequence was synthesized by Fmoc-based
SPPS on either an Applied Biosystems 433A or a Pioneer automated
peptide synthesizer using CLEAR-Acid resin. It was then cleaved
from the resin using 4.0 mL of freshly-prepared, Reagent K37 (TFA/
water/thioanisole/phenol/ethanedithiol 82.5:5:5:5:2.5) for 2.5 h,
precipitated with 100.0 mL of anhydrous ether, and centrifuged to

5682
K. Kyro et al. / Bioorg. Med. Chem. 18 (2010) 5675–5684
form a pellet. The pellet was washed with ether, solubilized in a
small amount of DMF, diluted in 0.1% aqueous TFA, and filtered by
vacuum filtration. The crude material was purified by RP-HPLC using
0.1% TFA/H2O (solvent A) and 0.1% TFA/CH3CN (solvent B) with the
following elution profile performed at 5.0 mL/min: 5.0 min 0% sol-
vent B; linear gradient from 0% to 60% solvent B over 120 min. The
solvent mixture was removed by lyophilization to afford 20.0 mg
(30%) of 1. Purity by RP-HPLC: 94.4%, tR = 41 min., ESI-MS (m/z):
[M+H]+ calcd for C58H94N16O17S2 1351.7236, found 1351.4039;
[M+2H]2+ calcd for C58H95N16O17S2 676.3212, found 676.3596;
[M+3H]3+ calcd for C58H96N16O17S2 451.2141, found 451.2424.
2 h of reaction time, 200 lL of BME was added (1.43 M) to quench
the reaction and reverse any sulfonium salt formation.29 It was stir-
red in BME 2.5 h, filtered, and diluted with 0.1% aqueous TFA before
RP-HPLC purification. The crude was purified by RP-HPLC using 0.1%
TFA/H2O (solvent A) and 0.1% TFA/CH3CN (solvent B) and the follow-
ing elution profile performed at 2.5 mL/min: 5.0 min 0% solvent B;
linear gradient from 30% to 100% solvent B over 140 min. The solvent
mixture was removed by lyophilization to afford 12 mg (74%) of 6.
Purity by RP-HPLC: 97.0%, tR = 61 min., ESI-MS (m/z): [M+2Na]2+
calcd for C110H171N21O28S3Na2 1188.0780, found 1187.8760;
[M+2Na+H]3+ calcd for C110H172N21O28S3Na2 792.3853, found
792.2635; [M+2Na+2H]4+ calcd for C110H173N21O28S3Na2 594.5390,
4.2.2. Synthesis of Abz-KSKTKC(C5BP)K(Dnp)IM, 4
found 594.4493. UV
e .
349 = 18,000 mMꢁ1 cmꢁ1
Peptide 1 (10.0 mg, 7.0
of 2:1:1 DMF/n-butanol/0.1% aqueous TFA (v/v/v) and (E)-4-(30-ben-
zoylbenzyloxy)-3-methyl-2-buten-1-bromide, 3 (10.0 mg, 30 mol,
4.0 equiv), synthesized according to published methods,15,19 was
then added. Zn(OAc)2 (8.0 mg, 40 mol, 5.0 equiv) was dissolved
lmol, 1.0 equiv) was dissolved in 10.0 mL
4.3. Kinetic analysis
l
4.3.1. In vitro fluorescence-based Ca1a2X proteolysis assay
Carbonate-washed membrane fractions containing yeast Rce1p
were used as the source of enzyme. Membranes were isolated
according to reported methods and prepared as 1.0 mg/mL total
protein stock solutions in lysis buffer (50 mM Tris, pH 7.5, 0.2 M
sorbitol, 1 mM EDTA, 0.2% NaN3) containing a protease inhibitor
l
in a small amount 0.1% aqueous TFA and added to the reaction.
The reaction was monitored by RP-HPLC and was complete in 2 h.
It was then diluted with 0.1% aqueous TFA before RP-HPLC
purification. The crude material was purified by RP-HPLC using
0.1% TFA/H2O (solvent A) and 0.1% TFA/CH3CN (solvent B) and the
following elution profile performed at 5.0 mL/min: 5.0 min 0% sol-
vent B; linear gradient from 0% to 60% solvent B over 120 min. The
solvent mixture was removed by lyophilization yielding 3.3 mg
(27%) of 4. Purity by RP-HPLC: 91.5%, tR = 60 min., ESI-MS (m/z):
[M+2H]2+ calcd for C77H113N16O19S2 815.3866, found 815.3873;
[M+3H]3+ calcd for C77H114N16O19S2 543.9244, found 543.9283. UV
cocktail (1 lg/mL each chymostatin, leupeptin, pepstatin;
0.85
l
g/mL aprotinin; 1 mM PMSF) and stored at ꢁ80 °C.8,30 The
membranes were diluted with an equal volume of assay buffer
(100 mM Hepes, pH 7.5, 5 mM MgCl2) to 0.5 mg/mL and preincu-
bated in a metallic block for 10 min at 30 °C. Substrates 2 and 4
were serially diluted in assay buffer from their stock concentra-
tions (100 and 90
lM, respectively) into a 96-well V-shaped micro-
e
349 = 18,000 mMꢁ1 cmꢁ1
.
titer plate (three 15 l
L replicates) and transferred to 384-well
black flat bottom microtiter plate; 10 concentration points for each
substrate. The plate was covered with a lid and incubated 10 min
4.2.3. Synthesis of biotin-K(Dde)KSKTKCK(Dnp)IM, 5
The linear peptide sequence was synthesized by Fmoc-based
SPPS on a peptide synthesizer using CLEAR-Acid resin. The final
Fmoc protecting group was selectively removed from the protected
at 30 °C. Assays were initiated with the addition of 15 lL of diluted
membranes to the diluted substrates in the wells, resulting in a fi-
nal membrane protein concentration of 0.25 mg/mL, substrate con-
peptide resin (121.0 mg, 40
l
mol, 1.0 equiv) by tumbling it 15 min
centrations ranging 0–50
lM (2), or 0–45 lM (4), and a total assay
in a 1:4 (v/v) mixture of piperidine in DMF (5.0 mL), followed by
volume of 30 L. The fluorescence in the samples was measured
l
washing with DMF (2 ꢀ 5.0 mL) and methylene chloride
every 30–60 s for 1 h at 30 °C with 5 s shaking before each reading
at 340/420 nm excitation/emission wavelengths on a BioTek Syn-
ergy™ HT microplate fluorometer.
(1 ꢀ 5.0 mL). TFP–PEG3–Biotin (100 mg, 140
lmol, 4.0 equiv dis-
solved in 1.0 mL DMF) was added and it was coupled to the N-ter-
minus using DIEA (49.0 L, 300 mol, 8.0 equiv) and tumbling
l
l
48 h. The resin was then washed with DMF (2 ꢀ 5.0 mL) and meth-
ylene chloride (4 ꢀ 5.0 mL) and dried in vacuo. The peptide was
cleaved from the resin using 4.0 mL of freshly-prepared, Reagent
K (TFA/water/thioanisole/phenol/ethanedithiol 82.5:5:5:5:2.5) for
2.5 h, precipitated with anhydrous ether (100.0 mL), and centri-
fuged to form a pellet. The pellet was washed with ether, solubi-
lized in a small amount of DMF, diluted in 0.1% aqueous TFA, and
filtered by vacuum filtration. The crude product was purified by
RP-HPLC using 0.1% TFA/H2O (solvent A) and 0.1% TFA/CH3CN (sol-
vent B) with the following elution profile performed at 5.0 mL/min:
5.0 min 0% solvent B; linear gradient from 0% to 60% solvent B over
120 min. The solvent mixture was removed by lyophilization to af-
ford 20 mg (28%) of 5. Purity by RP-HPLC: 75.6%, tR = 52 min., ESI-
MS (m/z): [M+2Na]2+ calcd for C91H153N21O26S3Na2 1049.0127,
found 1048.6907; [M+2Na+H]3+ calcd for C91H154N21O26S3Na2
699.6751, found 699.4679; [M+2Na+2H]4+ calcd for C91H155N21
O26S3Na2 525.0063, found 524.5923.
4.3.2. Kinetic analysis
Data from experiments was collected as replicates (two for 2
and five for 4) and analyzed using GraphPad Prism 4.0. Data was
fit to the four parameter Hill Equation Y ¼ Vmax ꢂ Xh=ðKMh þ XhÞ
without constraints after providing approximate initial values.
Prior to analysis, the data (RFUsample) was corrected to take into ac-
count the observation that RFU and product formation are propor-
tional but do not have a 1:1 relationship. In part, this is due to
intermolecular quenching effects that increase as substrate con-
centration increases. A standard curve was created using trypsin
to determine the maximum fluorescence obtainable at various
substrate concentrations (RFUtrypsin). Both 2 and 4 contain trypsin
cleavage sites. A graph of RFUtrypsin versus substrate concentration
was plotted for each substrate and a best-fit line determined (2,
Y = 116.29X, R2 = 0.993; 4, Y = 168.23X, R2 = 0.9834). The equations
of the curves were used to extract the actual product concentration
for partially reacted samples (RFUsample). These values were used
for Prism analysis.
4.2.4. Synthesis of biotin-K(Dde)KSKTKC(C5BP)K(Dnp)IM, 6
Peptide 5 (15.0 mg, 7.0
l
mol, 1.0 equiv) was dissolved in 2.0 mL
4.4. Mass spectrometric analysis
of 2:1:1 DMF/n-butanol/0.1% aqueous TFA (v/v/v) and (E)-4-(30-
benzoylbenzyloxy)-3-methyl-2-buten-1-bromide, 3 (10.5 mg, 30
l
4.4.1. Large-scale proteolysis reactions for product structure
identification
mol, 4.0 equiv), was added. Zn(OAc)2 (8.0 mg, 40 lmol, 5.0 equiv)
was dissolved in a small amount 0.1% aqueous TFA and added to
Stock concentrations of 2 and 4 were diluted in Hepes buffer
the reaction. The reaction was monitored by RP-HPLC. Following
(100 mM Hepes, 5 mM MgCl2, pH 7.5) to 150 lM. Rce1p-contain-

K. Kyro et al. / Bioorg. Med. Chem. 18 (2010) 5675–5684
5683
ing membranes were then added to a final concentration of 0.3 mg/
mL (total protein). The samples were mixed briefly and incubated
in a 30 °C water bath for 3–3.5 h. The crude product was purified
using Sep-Pak reversed phase C18 columns equilibrated in solvent
A (0.1% TFA/H2O). The reaction mixtures containing proteolyzed
peptide were applied to the columns and fractions were eluted
with a stepwise gradient from 0% to 100% B using 4–6 mL each
mixture of solvents A and B. The individual fractions were evalu-
ated by ESI-MS, which showed the doubly-charged parent ions of
the products of cleavage in the 30%, 40%, and 50% B fractions. These
fractions were lyophilized separately and re-dissolved in solvent A
before MS analysis.
using enhanced chemiluminescence (ECL) on autoradiographic
film.
4.5.2. Competition experiments using 2 and 6
Photolysis reactions were conducted as described in Section 4.5.
All reactions (200
7.5, 0.75 M 6, and 0.25
pound 2 was used at 50
l
L) contained 100 mM Hepes, 5 mM MgCl2, pH
g/ L membranes containing Rce1p. Com-
M and 100 M in the assays evaluating
l
l l
l
l
competition. Pull-down and Western blotting were performed as
described in section 4.5.1.
Acknowledgments
4.4.2. MS–MS analysis of farnesylated and benzophenone-
modified substrates following enzymatic processing by Rce1p
The lyophilized samples from proteolysis of 2 and 4 were dis-
This research was supported by the National Institutes of
Health Grants GM58442 (M.D.D.), and GM067092 (W.K.S.). Some
equipment and materials were graciously supplied by Edgewood
Chemical Biological Center. Additional thanks to Bruce Witthuhn
at University of Minnesota Center for Mass Spectrometry and
Proteomics.
solved in 10
0.1% FA/CH3CN prior to MS analysis. MS was performed using
10 L injections (CI 25-32) and 5 min. data acquisition on a QSTAR
Pulsar quadrupole-TOF (time-of-flight) mass spectrometer
lL of 0.1% aqueous TFA and further diluted 2:50 in
l
i
equipped with a turbo ionspray source. The doubly-charged
[M+2H]2+ species (m/z = 509.3 and m/z = 546.4, respectively) for
the predicted products 7 and 8 were most abundant in the 40% B
fractions and were selected for subsequent MS–MS analysis. MS re-
sults were evaluated using Analyst software.
Supplementary data
Supplementary data (RP-HPLC chromatograms for 1, 4, 5, and 6
and summary tables of mass spectral data for 2, 4, 7, and 8) asso-
ciated with this article can be found, in the online version, at
doi:10.1016/j.bmc.2010.06.024.
4.5. Photoaffinity labeling
References and notes
All photolysis reactions were conducted at 4 °C in a UV Rayonet
photoreactor (Model # RPR-100, Southern New England Ultraviolet
Co.) equipped with sixteen RPR-2537 Å lamps and a circulating plat-
form that allows up to 13 samples to be irradiated simultaneously.
1. Dolence, J. M.; Steward, L. E.; Dolence, E. K.; Wong, D. H.; Poulter, C. D.
Biochemistry 2000, 39, 4096.
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All reactions (135
(10 ꢀ 43 mm) and contained 100 mM Hepes, 5 mM MgCl2, pH 7.5,
15 M 6, and 0.2 g/ L membranes containing or lacking Rce1p.
The reactions were photolyzed for 30 min using the apparatus de-
scribed above and quenched by flash-freezing in N2 (l).
lL) were performed in silanized quartz test tubes
l
l l
4.5.1. Pull-down of photolabeled Rce1p on streptavidin beads
followed by Western blot analysis
Irradiated samples were thawed and concentrated using protein
precipitation. The concentrated samples were solubilized in RIPA
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l
L; 150 mM NaCl, 50 mM TrisꢃHCl, 1.0% (w/v) sodium
deoxycholate, 1.0% (v/v) Triton X-100] containing 1.5% (w/v) SDS
and added to spin columns containing streptavidin agarose resin
(0.1–0.5 lg of protein/lL resin pre-equilibrated with RIPA/1.5%
SDS) and incubated 1–1.5 h at room temperature. Following brief
centrifugation, the resin was washed five times with RIPA/1.5% SDS.
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The samples were eluted by adding 2ꢀ sample buffer [15
lL;
4.0% SDS, 20% glycerol (v/v), 125 mM TrisꢃHCl (pH 6.8), 10%
2-mercaptoethanol (v/v), 0.004% bromophenol blue (w/v)] to each
spin column, heating to 95 °C for 5 min followed by centrifugation,
and repeated for a total of 30
lL of sample. The samples were then
combined with an additional 30
SDS–PAGE (12% Tris–glycine gels) electrophoresis, and transfer to
l
L of 2ꢀ sample buffer before
PVDF membranes.
The PVDF membranes were blocked with 5% (w/v) casein in
TBST [25 mM Tris, 150 mM NaCl, 0.1% Tween-20 (v/v), pH 7.4]
for 1 h, then incubated in 2.5% (w/v) casein in TBST containing
anti-HA antibody [mAb HA.11 (HA, 16B12, flu tag), 1:10,000 (Cov-
ance)] overnight at 4 °C. The membranes were then washed with
TBST and PBS [137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4,
2 mM KH2PO4] and incubated with 2.5% (w/v) casein in TBST con-
taining secondary antibody [ECL anti-mouse IgG, horseradish per-
oxidase-linked (NA931, from sheep), 1:10,000 (GE Healthcare)] for
2 h at room temperature. Membranes were washed and visualized
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