Design and Synthesis of Activity-Based Probes and Inhibitors for Bleomycin Hydrolase

Article abstract of DOI:10.1016/j.chembiol.2015.07.010

Summary Bleomycin hydrolase (BLMH) is a neutral cysteine aminopeptidase that has been ascribed roles in many physiological and pathological processes, yet its primary biological function remains enigmatic. In this work, we describe the results of screening of a library of fluorogenic substrates to identify non-natural amino acids that are optimally recognized by BLMH. This screen identified several substrates with k_cat/K_M values that are substantially improved over the previously reported fluorogenic substrates for this enzyme. The substrate sequences were used to design activity-based probes that showed potent labeling of recombinant BLMH as well as endogenously expressed BLMH in cell extracts, and in intact cells. Importantly, we identify potent BLMH inhibitors that are able to fully inhibit endogenous BLMH activity in intact cells. These probes and inhibitors will be valuable new reagents to study BLMH function in cellular and animal models of human diseases where BLMH is likely to be involved.

Full text of DOI:10.1016/j.chembiol.2015.07.010

Brief Communication
Design and Synthesis of Activity-Based Probes and
Inhibitors for Bleomycin Hydrolase
Graphical Abstract
Authors
Wouter A. van der Linden, Ehud Segal,
Matthew A. Child, Anna Byzia, Marcin
Dra˛ g, Matthew Bogyo
Correspondence
mbogyo@stanford.edu
In Brief
Bleomycin hydrolase is a neutral cysteine
aminopeptidase that has been ascribed
roles in many physiological and
pathological processes, but its primary
biological function remains enigmatic.
van der Linden et al. describe the
synthesis and evaluation of activity-
based probes, irreversible inhibitors, and
fluorogenic substrates for bleomycin
hydrolase.
Highlights
d
d
d
Application of substrate screening to identify bleomycin
hydrolase-specific scaffolds
Identification of cell-permeable irreversible inhibitors for
bleomycin hydrolase
Identification of cell-permeable activity-based probes for
bleomycin hydrolase
van der Linden et al., 2015, Chemistry & Biology 22, 995–1001
August 20, 2015 ª2015 Elsevier Ltd All rights reserved
http://dx.doi.org/10.1016/j.chembiol.2015.07.010

Chemistry & Biology
Brief Communication
Design and Synthesis of Activity-Based Probes
and Inhibitors for Bleomycin Hydrolase
Wouter A. van der Linden,1 Ehud Segal,1 Matthew A. Child,1 Anna Byzia,2 Marcin Dra˛ g,2 and Matthew Bogyo1,
*
1Departments of Pathology and Microbiology and Immunology, Stanford University School of Medicine, 300 Pasteur Drive, Stanford,
CA 94305, USA
2Division of Bioorganic Chemistry, Faculty of Chemistry, Wroclaw University of Technology, Wyb. Wyspianskiego 27, 50-370 Wroclaw,
Poland
*Correspondence: mbogyo@stanford.edu
http://dx.doi.org/10.1016/j.chembiol.2015.07.010
SUMMARY
the skin (Kamata et al., 2009), and therefore plays an important
role in maintaining epidermal integrity (Kamata et al., 2011).
Bleomycin hydrolase (BLMH) is a neutral cysteine BLMH has also been shown to play a role in peptide trimming
downstream of the proteasome, and thus has a role in the
production of peptides for antigen presentation (Stoltze et al.,
aminopeptidase that has been ascribed roles in
many physiological and pathological processes, yet
its primary biological function remains enigmatic. In
this work, we describe the results of screening of a
library of fluorogenic substrates to identify non-natu-
ral amino acids that are optimally recognized by
BLMH. This screen identified several substrates
with kcat/KM values that are substantially improved
2000; Kim et al., 2009); however, this role seems to be redundant
(Towne et al., 2007).
In addition to its aminopeptidase activity, BLMH has the ability
to hydrolyze homocysteine lactone, a reactive metabolite pro-
duced from methionine, which causes protein damage and
hyperhomocysteinemia and is implicated in multiple human
diseases, including Alzheimer’s disease. BLMH is implicated in
protection against homocysteine toxicity (Zimny et al., 2006;
Borowczyk et al., 2012). However, recent data showing that
over the previously reported fluorogenic substrates
for this enzyme. The substrate sequences were
used to design activity-based probes that showed another enzyme exists with higher homocysteine lactonase ac-
tivity has called into question the role for BLMH in homocysteine
detoxification (Marsillach et al., 2014). BLMH polymorphisms are
potent labeling of recombinant BLMH as well as
endogenously expressed BLMH in cell extracts,
and in intact cells. Importantly, we identify potent
BLMH inhibitors that are able to fully inhibit endoge-
nous BLMH activity in intact cells. These probes and
inhibitors will be valuable new reagents to study
BLMH function in cellular and animal models of hu-
man diseases where BLMH is likely to be involved.
also associated with sporadic Alzheimer’s disease (Montoya
et al., 1998; Papassotiropoulos et al., 2000). Ectopic expression
of BLMH increases processing of amyloid precursor, suggesting
a regulatory role for BLMH in the secretion of amyloid precursor
protein and b-amyloid, which are major components of Alz-
heimer’s disease-associated plaques (Lefterov et al., 2000,
2001). However, other studies have shown reduced homocyste-
ine lactonase activity in brains of Alzheimer patients that corre-
lated with a reduction in BLMH levels, thus suggesting a protec-
tive role for BLMH (Suszynska et al., 2010).
INTRODUCTION
At present, BLMH aminopeptidase activity has only been
¨
Bleomycin hydrolase (BLMH) is a cysteine aminopeptidase that measured using fluorogenic substrates (Bromme et al., 1996;
¨
is ubiquitously expressed in mammalian tissue (Bromme et al., Zimny et al., 2006). While these substrates provide a relatively
1996). BLMH was initially discovered for its ability to inactivate rapid and simple readout of enzyme activity, the resulting data
bleomycin (Umezawa et al., 1972; Schwartz et al., 1999), a are often difficult to interpret because other aminopeptidases
drug used extensively to treat cancer. BLMH is a cytosolic are likely to be active toward the reported substrates (Rut
neutral protease with a barrel-like structure composed of six et al., 2015). Activity-based probes circumvent this problem by
¨
monomers of 50 kDa each (Bromme et al., 1996; O’Farrell
covalently attaching to target enzymes, allowing direct identifi-
et al., 1999). The active sites of BLMH are located within the bar- cation and quantification of enzyme activity (Sanman and Bogyo,
rel (Hibino et al., 2013). After expression, the C terminus of the 2014). Furthermore, by screening substrate libraries of increased
protein undergoes self-cleavage yielding an enzyme with diversity, it should be possible to identify sequences that are
broad-specificity aminopeptidase activity (Joshua-Tor et al., optimized for BLMH and not cleaved by other aminopeptidases.
1995; Zheng et al., 1998). While the physiological roles of
In this article, we present a screen of a diverse substrate library
BLMH remain obscure, it has been suggested to be important made up of both natural and non-natural amino acids to identify
in several physiological and pathological processes. BLMH null optimal binding elements for BLMH. Using this approach we
mice have reduced neonatal survival, brain pathologies (Mon- were able to design selective substrates, activity-based probes,
toya et al., 2007), and a dermatitis phenotype. BLMH is involved and inhibitors for BLMH. These reagents can be used for
in the production of free amino acids as moisturizing agents in biochemical studies of the purified enzyme as well as to monitor
Chemistry & Biology 22, 995–1001, August 20, 2015 ª2015 Elsevier Ltd All rights reserved 995

Figure 1. Screening of a Diverse Fluoro-
genic Substrate Library
(A) Diagram of the fluorogenic substrate library
screening approach.
(B) Ranking of the top BLMH substrates ordered by
k
cat/KM values. The substrates containing Lys(2-
Cl-Cbz) and Cys(Bn) are effective, with activities
that are substantially higher than the best natural
amino acid.
then chloroacetylated the amine group in
1 and performed a subsequent substitu-
tion reaction to generate azide 2. Oxida-
tion of the thioether yielded sulfone 3,
which can be used as a general reagent
to make vinyl sulfone probes with a Click
handle. We converted commercially avail-
able Boc-Lys(2-Cl-Cbz)-OH (4a) and Boc-
Cys(Bn)-OH (5a) to the corresponding
Weinreb amides and reduced them to
their respective aldehydes (4c and 5c),
and reacted in the HWE olefination reac-
tion to yield the azide-labeled phenyl vinyl
sulfone inhibitors 4d and 5d. We obtained
the final Cy5 modified activity-based
probes WL1256 and WL1259 using Click
chemistry (Figure 2A).
and inhibit the endogenous protease target in cellular extracts,
intact cells, and potentially whole organisms.
To test the newly synthesized probes we incubated recombi-
nant BLMH (rBLMH) with increasing concentrations of each
probe, then measured labeling by SDS-PAGE analysis followed
by scanning of the gel for fluorescent-labeled protein (Figure 2B).
The labeling confirmed that both probes efficiently labeled the
RESULTS
We screened a hybrid tailored amino acid substrate library that recombinant protein, as indicated by the presence of a doublet
was recently described and used to find highly efficient sub- of 52 kDa corresponding to the expected size of rBLMH. The
strates of a number of aminopeptidases (Drag et al., 2010; Rut appearance of multiple labeled species is likely due to autopro-
et al., 2015). This library is made up of a diverse set of natural cessing of BLMH, as has been previously described (Zheng
and non-natural amino acids linked to a fluorogenic reporter et al., 1998). We next tested the limit of sensitivity of the probes
that provides a signal when the substrate is cleaved by a prote- by labeling with a set probe concentration (1 mM) and decreasing
ase. Because bleomycin hydrolase is an aminopeptidase, we the amount of the rBLMH in the labeling reaction (Figure 2C).
screened the library of single amino acid-ACC (7-amino-4-car- Ultimately, the probe WL1259 showed the most potent labeling
bamoylmethylcoumarin) substrates against the recombinant of the target, and was therefore used for validation studies tar-
protease (Figure 1A). Interestingly, this screen identified non-nat- geting the endogenously expressed enzyme.
ural amino acid-containing substrates that had kcat/KM values
To confirm that our optimal probe WL1259 was a viable tool for
greater than the best natural amino acid, methionine (Figure 1B). the study of BLMH function, we performed probe labeling
The top two substrates that we chose for further development studies in lysates from fibroblasts derived from wild-type (WT)
into inhibitors and active site probes were Lys(2-Cl-Cbz)-ACC and BLMH knockout (KO) mice (Figure 2D). Importantly, these
and S-benzylated cysteine.
results confirmed that the probe labeled a protein of the ex-
To design activity-based probes, we choose electrophiles that pected size of 52 kDa in WT lysate that was confirmed to be
would covalently label the active site nucleophile, but that also al- native BLMH due to its absence in the KO cell lysate. We
lowed incorporation of a tag that would not interfere with the free observed similar results when the probe was used to label intact
amino group required for aminopeptidase recognition. Therefore, fibroblast cells derived from WT and BLMH KO mice (Figure 2E).
we initially used the vinyl sulfone, since this electrophile has been These data confirmed that the probe was able to enter cells and
extensively applied to probes of cathepsins and also the protea- label the native BLMH.
some (Verdoes et al., 2006; Yuan et al., 2006). We synthesized a
Given the success of the activity-based probes in both lysates
reagent that allows introduction of a vinyl sulfone equipped with and intact cells, we used the same general scaffolds to generate
an azide via the Horner-Wadsworth-Emmons (HWE) reaction inhibitors of BLMH that could be used to block its function in vivo.
(Figure 2A). Diethyl (iodo)methylphosphonate was reacted with We initially synthesized phenyl vinyl sulfone derivatives of
4-aminothiophenol sodium salt to yield aminothioether 1. We Lys(2-Cl-Cbz) and Cys(Bn), as these most closely matched the
996 Chemistry & Biology 22, 995–1001, August 20, 2015 ª2015 Elsevier Ltd All rights reserved

Figure 2. Synthesis and Evaluation of BLMH Probes Based on the Library Screening Result
(A) Synthesis scheme for the azide-intermediate and final Cy5-labeled probes.
(B) Labeling of 1 mg rBLMH over a range of probe concentrations. Samples were analyzed by SDS-PAGE followed by scanning of the gel using a flatbed laser
scanner to detect the Cy5 signal.
(C) Labeling of decreasing amounts of rBLMH using 1 mM of each of the two primary Cy5 probes.
(D) Assessment of BLMH labeling by the selected probe, WL1259 in wild-type (WT) and BLMH knockout (KO) fibroblast lysates. rBLMH is shown in the first lane as
a standard, but appears as a slightly higher molecular weight than the native enzyme due to the presence of HIS6 tag.
(E) Intact WT and BLMH KO cells were incubated with WL1259 (10 mM) for 3 hr at 37ꢀC, then cells were lysed and analyzed by SDS-PAGE as in (D).
The location of BLMH is indicated by a star and is only visible in the WT cells treated with the probe.
activity-based probes WL1256 and WL1259. We also synthe- cysteine proteases (Powers et al., 2002; Kato et al., 2005; Deu
sized the methyl sulfone versions of the compounds to see et al., 2010). The synthesis of all the potential BLMH inhibitors
whether the smaller methyl group would reduce steric hindrance is shown in Figure 3A (Wang et al., 2004). To measure the inhib-
in the active site and result in greater potency. Both classes of itory potencies of the inhibitors, we used a fluorogenic substrate
vinyl sulfone compounds were generated using the same chem- assay with the reported BLMH substrate Met-AMC and rBLMH
istry as described for the probe synthesis (Bogyo et al., 1997). (Figure 3B). We found that while the original vinyl sulfone com-
We also synthesized the acyloxymethyl ketone (AOMK) and phe- pounds had overall good potencies, the AOMK and PMK were
noxymethyl ketone (PMK) version of the lead compounds, as more potent inhibitors of BLMH by several orders of magnitude
these two electrophiles have been extensively used to target (Figure 3C). These data confirm that our chosen scaffold can
Chemistry & Biology 22, 995–1001, August 20, 2015 ª2015 Elsevier Ltd All rights reserved 997

Figure 3. Synthesis and Evaluation of BLMH Inhibitors
(A) Synthesis of six inhibitors for BLMH containing multiple different cysteine-reactive electrophiles.
(B) Fluorogenic substrate assay using rBLMH to determine the IC50 of inhibitors. Fluorogenic substrate experiments were performed in triplicate for each point
and were normalized. Error bars represent SEM.
(C) Calculated IC50 and ki values for the BLMH inhibitors. ki values were derived from curve fitting of the normalized data and are presented as the average SD.
998 Chemistry & Biology 22, 995–1001, August 20, 2015 ª2015 Elsevier Ltd All rights reserved

Figure 4. Inhibition of Endogenous BLMH in Intact Cells Using the Optimized Inhibitors
Embryonic fibroblast cells derived from wild-type (WT) and BLMH knockout (KO) mice were incubated with inhibitors at the indicated concentrations for 3 hr at
37ꢀC. Cells were washed and lysed, and the lysate incubated with 1 mM WL1259 for 1 hr at 37ꢀC. Samples were analyzed by SDS-PAGE followed by scanning of
the gel by a flatbed laser scanner to observe fluorescence. The box indicates the location of native BLMH that is absent in the knockout (KO) cells.
be used to yield highly potent inhibitors that are effective in the used to shed light on its still enigmatic primary biological
low nanomolar concentration range.
functions.
As a final test of potency and cell permeability, we treated
intact mouse embryonic fibroblasts (MEF) or BLMH KO mouse
fibroblasts with a range of doses of each inhibitor. We then lysed
cells and labeled them with WL1259, and measured residual
activity of the native BLMH enzyme by SDS-PAGE analysis (Fig-
ure 4). We found that all of the vinyl sulfones were able to pene-
trate cells and completely inhibit BLMH, but only at micromolar
concentrations, consistent with the measured potencies of
the compounds against the recombinant enzyme. The AOMK
(WL911) and PMK (WL920) derivatives, on the other hand,
were able to completely block activity of the native BLMH at
mid to high nanomolar concentrations. Thus, we have identified
a class of highly potent inhibitors of this enigmatic protease that
can be used on intact cells to block enzyme activity and allow
studies of protease function.
EXPERIMENTAL PROCEDURES
Synthesis of Inhibitors and Probes
Detailed methods and compound characterization for all inhibitors and activ-
ity-based probes can be found in the Supplemental Materials and Methods
section.
Cloning, Expression, and Purification of rBLMH
Details regarding cloning, expression, and purification of rBMLH can be found
in the Supplemental Information.
Screening of Fluorescent Substrates
BLMH was assayed in 100 mM Tris-HCl (pH 7.5), 1 mM EDTA, and 1 mM DTT.
Assays were performed at 37ꢀC and enzyme was incubated at 37ꢀC for
30 min before adding substrate. Screening of the library was carried out at
2 mM substrate concentration, with 20 nM enzyme. Release of fluorophore
was monitored continuously with excitation at 355 nm and emission at
460 nm for 30–45 min, and the linear portion of the progress curve was
used to calculate velocity. All experiments were repeated at least three times.
Analysis of the results was based on total relative fluorescence units for each
substrate, setting the highest value to 100% and adjusting the other results
accordingly.
SIGNIFICANCE
Although BLMH has been studied for many years, chemical
tools to study its function have not been reported. Here,
we describe activity-based probes and potent cell-perme-
able inhibitors of BLMH. Given that this enzyme has been
postulated to be involved in many physiological processes
important in human diseases, such as antigen processing,
homocysteine lactone detoxification, and Alzheimer’s dis-
ease, the inhibitors and probes presented here will be highly
valuable reagents for further study of BLMH, and can be
Determination of Kinetic Parameters kcat, KM, and kcat/KM
Enzyme assay conditions were as follows: 100-ml reaction with eight different
substrate concentrations. Release of ACC fluorophore was monitored as
above. Absolute ACC concentrations were calculated by the hydrolysis of
three independent ACC-coupled substrates at known concentration, and
average value was determined. Concentration of DMSO in the assay was
less than 1%.
Chemistry & Biology 22, 995–1001, August 20, 2015 ª2015 Elsevier Ltd All rights reserved 999

Fluorogenic Substrate Assay
REFERENCES
BLMH activity was measured in black 96-well plates (n = 3). rBLMH (1 nM) was
incubated with inhibitors (1003 DMSO stock) in 100 mM Tris (pH 7.5), 1 mM
EDTA, and 1 mM DTT for 1 hr at 37ꢀC. Substrate (100 mM Met-AMC) was
added, and 7-amino-4-methylcoumarin (AMC) fluorescence was monitored
every minute for 45 min at 37ꢀC using a Biotek plate reader. Half-maximal
inhibitory concentration (IC50) values were calculated using Graphpad Prism.
ki values were calculated using the formula v/v0 = exp(Àki[I]t).
Bogyo, M., McMaster, J.S., Gaczynska, M., Tortorella, D., Goldberg, A.L., and
Ploegh, H. (1997). Covalent modification of the active site threonine of protea-
somal b subunits and the Escherichia coli homolog HsIV by a new class of in-
hibitors. Proc. Natl. Acad. Sci. USA 94, 6629–6634.
ꢀ
Borowczyk, K., Tisonczyk, J., and Jakubowski, H. (2012). Metabolism and
neurotoxicity of homocysteine thiolactone in mice: protective role of bleomycin
hydrolase. Amino Acids 43, 1339–1348.
BLMH KO Mouse Generation
¨
Bromme, D., Rossi, A.B., Smeekens, S.P., Anderson, D.C., and Payan, D.G.
KO mice were bred from frozen heterozygous embryos obtained from Jack-
son. The deletion of the BLMH gene was confirmed by genotyping, and loss
of protein expression was confirmed by western blot. All animal care and
experimentation was conducted in accordance with current NIH and Stanford
University Institutional Animal Care and Use Committee guidelines.
(1996). Human bleomycin hydrolase: molecular cloning, sequencing, func-
tional expression, and enzymatic characterization. Biochemistry 35, 6706–
6714.
Deu, E., Leyva, M.J., Albrow, V.E., Rice, M.J., Ellman, J.A., and Bogyo, M.
(2010). Functional studies of Plasmodium falciparum dipeptidyl aminopepti-
dase I using small molecule inhibitors and active site probes. Chem. Biol.
17, 808–819.
Labeling Experiments in Cell Lysate
Mouse immortalized fibroblasts and mouse BLMH KO immortalized fibroblasts
were cultured on DMEM (Gibco) supplemented with 10% fetal calf serum
(Gibco), 100 units/ml penicillin, and 100 mg/ml streptomycin (Gibco) in a 5%
CO2 humidified incubator at 37ꢀC. Cells were harvested, washed twice with
PBS, and permeated in lysis buffer (50 mM Tris [pH 7.4], 150 mM NaCl,
20 mM MgCl2, 0.5% NP-40, 2 mM DTT) for 20 min on ice and centrifuged at
16,100relativecentrifugalforcefor20minat4ꢀC. Thesupernatantwascollected
and the protein content determined by bicinchoninic acid (BCA) assay (Pierce).
Total lysate (15 or 10 mg total protein) was incubated with the inhibitors (103 so-
lution in DMSO) for 1 hr at 37ꢀC. Reaction mixtures were boiled in Laemmli buffer
containing b-mercaptoethanol for 3 min before being resolved by 15% SDS-
PAGE. In-gel detection of fluorescently labeled proteins was performed directly
in the wet gel slabs on the Typhoon Variable Mode Imager (Amersham Biosci-
ences) using the Cy3/Tamra settings (lex 532 nm, lem 560 nm) for WL1189
and WL1192, or Cy5 settings (lex 650 nm, lem 670 nm) for WL1256 and WL1259.
Drag, M., Bogyo, M., Ellman, J.A., and Salvesen, G.S. (2010). Aminopeptidase
fingerprints, an integrated approach for identification of good substrates and
optimal inhibitors. J. Biol. Chem. 285, 3310–3318.
Hibino, T., Kamata, Y., and Takeda, A. (2013). Bleomycin hydrolase. In
Handbook of Proteolytic Enzymes, Third Edition, N.D. Rawlings and G.S.
Salvesen, eds. (Elsevier), pp. 1974–1980.
Joshua-Tor, L., Xu, H.E., Johnston, S.A., and Rees, D.C. (1995). Crystal struc-
ture of a conserved protease that binds DNA: the bleomycin hydrolase, Gal6.
Science 269, 945–950.
Kamata, Y., Taniguchi, A., Yamamoto, M., Nomura, J., Ishihara, K., Takahara,
H., Hibino, T., and Takeda, A. (2009). Neutral cysteine protease bleomycin hy-
drolase is essential for the breakdown of deiminated filaggrin into amino acids.
J. Biol. Chem. 284, 12829–12836.
Kamata, Y., Yamamoto, M., Kawakami, F., Tsuboi, R., Takeda, A., Ishihara, K.,
and Hibino, T. (2011). Bleomycin hydrolase is regulated biphasically in a differ-
entiation- and cytokine-dependent manner: relevance to atopic dermatitis?
J. Biol. Chem. 286, 8204–8212.
Labeling/Inhibition Experiments in Living Cells
Cells (50,000) were seeded and grown overnight. Stock solutions of inhibitors
or probes (1003) were added to 0.5 ml of medium and the cells were incubated
for 3 hr at 37ꢀC. Cells were harvested and washed twice with PBS, and lysate
was prepared as described above. The protein content was determined by
BCA assay (Pierce). For cells labeled with probe, the lysate was immediately
boiled in Laemmli sample buffer and resolved as described above. Lysates
(20 mg total protein, diluted with lysis buffer) from the inhibitor-treated cells
were labeled with 1 mM probe D for 1 hr at 37ꢀC, and boiled in Laemmli sample
buffer and resolved as described above. Staining of the gel with Coomassie
brilliant blue was used to confirm equal protein loading.
Kato, D., Boatright, K.M., Berger, A.B., Nazif, T., Blum, G., Ryan, C., Chehade,
K.A., Salvesen, G.S., and Bogyo, M. (2005). Activity-based probes that target
diverse cysteine protease families. Nat. Chem. Biol. 1, 33–38.
Kim, E., Kwak, H., and Ahn, K. (2009). Cytosolic aminopeptidases influence
MHC class I-mediated antigen presentation in an allele-dependent manner.
J. Immunol. 183, 7379–7387.
Lefterov, I.M., Koldamova, R.P., and Lazo, J.S. (2000). Human bleomycin
hydrolase regulates the secretion of amyloid precursor protein. FASEB J. 14,
1837–1847.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Materials and Methods and
can be found with this article online at http://dx.doi.org/10.1016/j.chembiol.
2015.07.010.
Lefterov, I.M., Koldamova, R.P., Lefterova, M.I., Schwartz, D.R., and Lazo, J.S.
(2001). Cysteine 73 in bleomycin hydrolase is critical for amyloid precursor
protein processing. Biochem. Biophys. Res. Commun. 283, 994–999.
Marsillach, J., Suzuki, S.M., Richter, R.J., McDonald, M.G., Rademacher,
P.M., MacCoss, M.J., Hsieh, E.J., Rettie, A.E., and Furlong, C.E. (2014).
Human valacyclovir hydrolase/biphenyl hydrolase-like protein is a highly effi-
cient homocysteine thiolactonase. PLoS One 9, e110054.
ACKNOWLEDGMENTS
We thank S.R. Lynch at the Stanford University Department of Chemistry NMR
facility for assistance, and Josh Lichtman and Josh Elias for recording HRMS
spectra. This work was supported by the NIH grant R01 EB005011 (to M.B.).
W.A.v.d.L. was supported by a Rubicon fellowship from the Netherlands Orga-
nization for Scientific Research (NWO). The Drag laboratory is supported by
the State for Scientific Research Grant N N401 042838 in Poland. The work
was also supported by a statutory activity subsidy from the Polish Ministry
of Science and Higher Education for the Faculty of Chemistry at Wroclaw Uni-
versity of Technology. We thank Marcin Poreba, Zofia Pilch, and Paulina Piatek
for help with synthesis of fluorogenic substrates.
Montoya, S.E., Aston, C.E., DeKosky, S.T., Kamboh, M.I., Lazo, J.S., and
Ferrell, R.E. (1998). Bleomycin hydrolase is associated with risk of sporadic
Alzheimer’s disease. Nat. Genet. 18, 211–212.
Montoya, S.E., Thiels, E., Card, J.P., and Lazo, J.S. (2007). Astrogliosis and
behavioral changes in mice lacking the neutral cysteine protease bleomycin
hydrolase. Neuroscience 146, 890–900.
O’Farrell, P.A., Gonzalez, F., Zheng, W., Johnston, S.A., and Joshua-Tor, L.
(1999). Crystal structure of human bleomycin hydrolase, a self-compartmen-
talizing cysteine protease. Structure 7, 619–627.
Received: June 9, 2015
Revised: July 2, 2015
Papassotiropoulos, A., Bagli, M., Jessen, F., Frahnert, C., Rao, M.L., Maier,
W., and Heun, R. (2000). Confirmation of the association between bleomycin
hydrolase genotype and Alzheimer’s disease. Mol. Psychiatry 5, 213–215.
Accepted: July 9, 2015
Published: August 6, 2015
1000 Chemistry & Biology 22, 995–1001, August 20, 2015 ª2015 Elsevier Ltd All rights reserved

Powers, J.C., Asgian, J.L., Ekici, O.D., and James, K.E. (2002). Irreversible
inhibitors of serine, cysteine, and threonine proteases. Chem. Rev. 102,
4639–4750.
Umezawa, H., Takeuchi, T., Hori, S., Sawa, T., and Ishizuka, M. (1972). Studies
on the mechanism of antitumor effect of bleomycin on squamous cell carci-
noma. J. Antibiot. 25, 409–420.
Rut, W., Kasperkiewicz, P., Byzia, A., Poreba, M., Groborz, K., and Drag, M.
(2015). Recent advances and concepts in substrate specificity determination
of proteases using tailored libraries of fluorogenic substrates with unnatural
amino acids. Biol. Chem. 396, 329–337.
Verdoes, M., Florea, B.I., Menendez-Benito, V., Maynard, C.J., Witte, M.D.,
van der Linden, W.A., van den Nieuwendijk, A.M., Hofmann, T., Berkers,
C.R., van Leeuwen, F.W., et al. (2006). A fluorescent broad-spectrum protea-
some inhibitor for labeling proteasomes in vitro and in vivo. Chem. Biol. 13,
1217–1226.
Sanman, L.E., and Bogyo, M. (2014). Activity-based profiling of proteases.
Annu. Rev. Biochem. 83, 249–273.
Wang, D., Schwinden, M.D., Radesca, L., Patel, B., Kronenthal, D., Huang,
M.H., and Nugent, W.A. (2004). One-carbon chain extension of esters to
a-chloroketones: a safer route without diazomethane. J. Org. Chem. 69,
1629–1633.
Schwartz, D.R., Homanics, G.E., Hoyt, D.G., Klein, E., Abernethy, J., and Lazo,
J.S. (1999). The neutral cysteine protease bleomycin hydrolase is essential for
epidermal integrity and bleomycin resistance. Proc. Natl. Acad. Sci. USA 96,
4680–4685.
¨
Stoltze, L., Schirle, M., Schwarz, G., Schroter, C., Thompson, M.W., Hersh,
Yuan, F., Verhelst, S.H., Blum, G., Coussens, L.M., and Bogyo, M. (2006). A se-
lective activity-based probe for the papain family cysteine protease dipeptidyl
peptidase I/cathepsin C. J. Am. Chem. Soc. 128, 5616–5617.
L.B., Kalbacher, H., Stevanovic, S., Rammensee, H.G., and Schild, H.
(2000). Two new proteases in the MHC class I processing pathway. Nat.
Immunol. 1, 413–418.
Zheng, W., Johnston, S.A., and Joshua-Tor, L. (1998). The unusual active site
of Gal6/bleomycin hydrolase can act as a carboxypeptidase, aminopeptidase,
and peptide ligase. Cell 93, 103–109.
Suszynska, J., Tisonczyk, J., Lee, H.G., Smith, M.A., and Jakubowski, H.
(2010). Reduced homocysteine-thiolactonase activity in Alzheimer’s disease.
J. Alzheimers Dis. 19, 1177–1183.
Towne, C.F., York, I.A., Watkin, L.B., Lazo, J.S., and Rock, K.L. (2007).
Analysis of the role of bleomycin hydrolase in antigen presentation and the
generation of CD8 T cell responses. J. Immunol. 178, 6923–6930.
Zimny, J., Sikora, M., Guranowski, A., and Jakubowski, H. (2006). Protective
mechanisms against homocysteine toxicity: the role of bleomycin hydrolase.
J. Biol. Chem. 281, 22485–22492.
Chemistry & Biology 22, 995–1001, August 20, 2015 ª2015 Elsevier Ltd All rights reserved 1001