Organelle-specific activity-based protein profiling in living cells

Article abstract of DOI:10.1002/anie.201309135

A multimodal activity-based probe for targeting acidic organelles was developed to measure subcellular native enzymatic activity in cells by fluorescence microscopy and mass spectrometry. A cathepsin-reactive warhead conjugated to a weakly basic amine and a clickable alkyne, for subsequent appendage of a fluorophore or biotin reporter tag, accumulated in lysosomes as observed by structured illumination microscopy (SIM) in J774 mouse macrophage cells. Analysis of in vivo labeled J774 cells by mass spectrometry showed that the probe was very selective for cathepsins B and Z, two lysosomal cysteine proteases. Analysis of starvation-induced autophagy, a catabolic pathway involving lysosomes, showed a large increase in the number of tagged proteins and an increase in cathepsin activity. The organelle-targeting of activity-based probes holds great promise for the characterization of enzyme activities in the myriad diseases linked to specific subcellular locations, particularly the lysosome. Basic targeting: Organelle-targeted chemoproteomics in live cells is possible through tuning the physicochemical properties of an activity-based probe. A weakly basic amine was used to target an activity-based probe to the lysosome.

Full text of DOI:10.1002/anie.201309135

Angewandte
Chemie
DOI: 10.1002/anie.201309135
Proteomics
Organelle-Specific Activity-Based Protein Profiling in Living Cells**
Susan D. Wiedner, Lindsey N. Anderson, Natalie C. Sadler, William B. Chrisler,
Vamsi K. Kodali, Richard D. Smith, and Aaron T. Wright*
Abstract: A multimodal activity-based probe for targeting
acidic organelles was developed to measure subcellular native
enzymatic activity in cells by fluorescence microscopy and
mass spectrometry. A cathepsin-reactive warhead conjugated
to a weakly basic amine and a clickable alkyne, for subsequent
appendage of a fluorophore or biotin reporter tag, accumu-
lated in lysosomes as observed by structured illumination
microscopy (SIM) in J774 mouse macrophage cells. Analysis
of in vivo labeled J774 cells by mass spectrometry showed that
the probe was very selective for cathepsins B and Z, two
lysosomal cysteine proteases. Analysis of starvation-induced
autophagy, a catabolic pathway involving lysosomes, showed
a large increase in the number of tagged proteins and an
increase in cathepsin activity. The organelle-targeting of
activity-based probes holds great promise for the character-
ization of enzyme activities in the myriad diseases linked to
specific subcellular locations, particularly the lysosome.
thus endeavored to analyze specific active enzyme popula-
tions in defined organelles within live cells by using cell-
permeable organelle-directed activity-based probes (ABP).
We envisioned an acidotropic ABP that would accumu-
late in acidic organelles for the interrogation of resident
enzyme activity. High molecular weight probes containing
fluorophores and/or enrichment tags are known, but they
require cell penetrating peptides or receptor motifs, in
addition to long labeling times, in order to enter live cells
and accumulate.[5] Smaller ABPs can reach their enzyme
target quickly, but any organelle accumulation is due to
enzyme localization rather than defined probe properties.[2b,6]
Our envisioned ABP requires a moiety with specific phys-
icochemical properties[7] and an alkyne for CuAAC-mediated
conjugation of a fluorophore or enrichment tag.
As a proof of concept, we designed a lysosome-targeting
ABP. Lysosomes are ubiquitous in mammalian cells, impor-
tant for cell homeostasis, and play a role in cancer, Alzheim-
erꢀs disease, and numerous orphan diseases.[8] Lysosomes are
acidic organelles (pH value < 5) that contain at least 60
soluble hydrolase enzymes. Lysosomal proteomics is increas-
ingly important for the study of lysosome-associated diseases
and the discovery of novel enzymes.[8] Lysosomes are targeted
by weakly basic amines and lipophilic moieties, which
accumulate in acidic environments owing to passive diffusion
of a neutral species across the membrane into an acidic milieu
where the weak base is protonated, thereby preventing
diffusion back across the membrane.[9]
A
ctivity-Based Protein Profiling (ABPP) utilizes targeted
chemical probes to determine enzyme activities in vitro,
in situ, or in vivo.[1] Employing probe derivatives that contain
an azide or alkyne moiety permits multiple applications of
ABPP, such as fluorescence microscopy and mass spectrom-
etry (MS), by direct attachment of reporting groups through
copper-catalyzed azide–alkyne cycloadditions (CuAAC).[2]
Small probe size increases cell permeability, thereby allowing
interrogation of the native proteome within intact cells. The
use of ABPP to irreversibly bind shared catalytic features of
soluble enzyme families within a defined organelle popula-
tion is an exceptional challenge for chemoproteomics.[3]
Organelle proteomics is plagued by difficulties in distinguish-
ing true resident proteins from copurifying contaminant
proteins, and cellular disruption for organelle isolation
potentially interferes with native enzymatic activity.[4] We
We targeted acidic organelles by incorporating readily
available 3-(2,4-dinitroanilino)-3’-amino-N-methyldipropyl-
amine (DAMP) into an ABP (Figure 1). DAMP is known to
rapidly accumulate in acidic organelles owing to its primary
amine and pKa value of approximately 10.[10] Furthermore,
the DAMP primary amine provides a handle to append 1) an
alkyne for CuAAC,[11] and 2) a broadly reactive inhibitor of
papain cysteine protease, ethyl succinate epoxide, which is
extensively used for labeling cathepsins.[2b,5,6,11] The synthesis
of organelle-targeting DAMP epoxide probes (DEX-1 and
DEX-2) is described in the Supporting Information (Figure 1
[*] Dr. S. D. Wiedner, L. N. Anderson, N. C. Sadler, W. B. Chrisler,
V. K. Kodali, Dr. R. D. Smith, Dr. A. T. Wright
Biological Sciences Division, Pacific Northwest National Laboratory
902 Battelle Blvd, Richland, WA 99352 (USA)
E-mail: aaron.wright@pnnl.gov
[**] This work was supported in part by the Laboratory Directed
Research and Development Program at PNNL, a multiprogram
national laboratory operated by Battelle for the U.S. DOE under
Contract DE-AC05-76L01830, and by the NIH NIGMS (8 P41
GM103493-11). S.D.W. was supported by the PNNL Linus Pauling
Distinguished Postdoctoral Fellowship. Work was performed in the
Environmental Molecular Sciences Laboratory, a US DOE-BER
national scientific user facility at PNNL. This work used instru-
mentation and capabilities developed under support from the NIH
(8 P41 GM103493-11) and the DOE-BER.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201309135.
Figure 1. DAMP-derived lysosome-targeting activity-based probes.
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and Figures S1 and S2 in the Supporting Information). An
ABP lacking DAMP (DEX-3) was synthesized as a weak-acid
negative control (Figure 1 and Figure S2).[9] We suspected
that disruption of an intramolecular hydrogen bond between
the basic amines of DAMP by incorporating the amide of
DEX-1 would prevent efficient accumulation in acidic
organelles; therefore, we synthesized the amine derivative
DEX-2.
We applied our suite of organelle-targeting probes to
A549 human epithelial cells and J774 mouse macrophage cells
(Figure 2). Imaging revealed DEX-2 accumulation in punc-
tate vesicles throughout the A549 and J774 cells, while DEX-3
and DEX-1 gave diffuse cell labeling (Figure 2). The more
basic DEX-2 accumulates in punctate vesicles quickly and
permanently (Figure S3), with little cytosolic labeling. All
ABPs react with similar nucleophilic proteases in A549 global
cell lysate (Figure S4A); additionally, comparison of in vivo
J774 labeling shows that DEX-1 and DEX-2 have similar
intracellular targets except for two intense bands of low
molecular weight (MW, < 30 kDa, Figure S4B). Selective
labeling of these low MW proteins and accumulation of DEX-
2 is thus dependent on the basic amine.
The accumulation of DEX-2 is dependent on the acidic
environment of the target organelle and the activity of the
target enzyme. Pretreatment of J774 with excess NH4Cl,
known to neutralize acidic environments, reduced the label-
ing intensity of the punctate vesicles (Figure S5). Pretreat-
ment of the cells with iodoacetamide (IAM) or ethyl (2S,3S)-
oxirane dicarboxylate also reduced the labeling intensity
(Figure S5). The fluorescence-imaging experiments are con-
sistent with gel-based analysis; pretreatment of J774 cells with
NH4Cl and a slight excess of the cathepsin inhibitor E-64d
suppresses labeling of the bands at 52 kDa, 31 kDa (*), and
27 kDa (*), thus suggesting that these proteins are cathepsins
and that labeling is dependent on the acidic environment
(Figure 3). The aspartyl protease inhibitor pepstatin A did not
suppress labeling. DEX-2 labels purified cathepsin B and
J774 proteins in a dose-dependent manner (Figure S6).
Notably, the intense band at approximately 40 kDa appears
to be the result of CuAAC (Figure S7).
Figure 3. SDS-PAGE fluorescence imaging of DEX-2 labeled proteins in
J774 cells following in vivo pretreatment with E-64d (58 mm), pepstatin
A (58 mm), or NH4Cl (30 mm) and then labeled with DEX-2 (37 mm).
The Coomassie protein stain (right-hand image) shows equivalent
protein loading of gel lanes. LC–MS analysis of J774 cells labeled
in vivo with DEX-2 confirms labeling of CatB (28 kDa) and CatZ
(27 kDa), which are indicated by asterisks.
To identify the site of DEX-2 accumulation, J774 cells
treated with DEX-2 were immunostained for LAMP1,
a lysosome-membrane marker (Figure 4A). Confocal laser
scanning microscopy showed partial colocalization of DEX-2
with LAMP1 (Pearsonꢀs correlation coefficient (PCC) =
0.595 Æ 0.082, M1 = 0.919 Æ 0.026, M2 = 0.958 Æ 0.014;[12] Fig-
ure 4A). In fact it appears that a LAMP1-positive membrane
ring encircles the fluorescence signal of DEX-2 (arrows,
Figure 4A).[13] This encapsulation of the probe signal by
LAMP1 can be visualized at a resolution below the diffraction
limit of light (< 200 nm) by using super-resolution structured
illumination microscopy (SIM; Figure 4B).[14] Z-stack imag-
ing revealed that this phenomenon is found all the way
through the vesicle (Figure 4B and Videos S1 and S2 in the
Supporting Information), thus demonstrating that the probe
is confined within the lysosome.
We performed high resolution LC–MS/MS[22] analysis of
J774 cells treated with DEX-2 to characterize enzyme targets
under two growth conditions. No-probe DMSO controls were
run in parallel to account for background labeling. We
identified six proteins in healthy
J774 cells (Table S1 in the Sup-
porting Information). Cathe-
psins B and Z were the most
abundant proteins measured.
J774 cells were also labeled
under
autophagy
whether
starvation-induced
to determine
lysosome-targeted
DEX-2 could report on
enzyme activity changes during
stress (Figure S8). Autophagy,
the digestion and recycling of
cellular components such as
organelles and intracellular pro-
teins,[15] is important for cell
homeostasis and response to
stress and has been identified
as playing a role in cancer and
Figure 2. A549 and J774 cells were incubated with the DEX probes (10 mm and 2 mm, respectively, 1 h),
fixed, and conjugated to AlexaFluor 488 through CuAAC. DEX-2 gives punctate vesicle staining in both
cell lines. DEX-3 and DEX-1 give diffuse labeling throughout the cell. The no-probe control corresponds to
DMSO treatment followed by CuAAC with the fluorophore. Scale bars: 20 mm.
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Angewandte
Chemie
in the native cellular environment. The small activity increase
we measured for the cathepsins correlates with the very short
starvation interval used to induce autophagy in this study and
the transient nature of autolysosomes in autophagic flux. It
also demonstrates that Cathepsin B enzymatic activity is
variable and contingent upon cellular conditions.
In conclusion, we developed a multimodal organelle-
targeting ABP that rapidly accumulates in punctate vesicles
owing to the physicochemical characteristics of a weakly basic
amine. Through super-resolution SIM, we provide evidence
that these punctate vesicles are lysosomes. The probe was also
used to measure enzyme activity changes upon the induction
of autophagy in macrophage cells. The direction of an ABP to
specific organelles through its physicochemical properties
should be applicable to other organelles. Additionally, switch-
ing the electrophilic warhead of a defined organelle-targeting
ABP would allow the targeting of other resident enzyme
families. We believe this approach could fuse organelle and
activity-based proteomics and that it could be widely appli-
cable for the analysis of biological systems.
Figure 4. Colocalization of DEX-2 (green) with LAMP1 (red) in J774
cells. The J774 cells were treated with DEX-2 (2 mm) for 1 h. The cells
were fixed and the probe was conjugated to AlexaFluor 488. The
lysosomes were stained by immunofluorescence with anti-LAMP1,
a goat anti-rabbit IgG–Alexafluor 647 antibody. A) Confocal laser scan-
ning microscopy shows the green DEX-2 signal surrounded by the red
LAMP1 signal (indicated by arrows). B) The boxed region of the
confocal image was analyzed further by enhanced-resolution SIM.
Shown are the vertical Z-plane images. Running from the top of the
compartment (Z=1) to the middle of the compartment (Z=6); each
image represents a 110 nm step.
Received: October 18, 2013
Revised: November 26, 2013
Published online: February 6, 2014
Keywords: activity-based probes · fluorescence microscopy ·
lysosome · mass spectrometry · proteomics
Alzheimerꢀs disease.[16] Studies have shown that the expres-
sion and/or efficiency of hydrolytic enzymes can change
during autophagy.[17] Furthermore, DAMP accumulates in
autolysosomes, a fused lysosome–autophagosome in which
degradation occurs.[18] By using the same data-filtering
criteria, DEX-2 labeled 44 proteins during autophagy
(Table S1). 17 of the 44 proteins have previously been
observed in autophagosomes under nitrogen-starvation-
induced autophagy in human MCF7-eGFP-LC cells by
stable isotope labeling by amino acids in cell culture
(SILAC) and protein correlation profiling analysis.[19]
Autophagy constitutes degradation of cellular proteins
and organelles in autolysosomes. Consistent with this, DEX-
2-labeled proteins in autophagic cells come from a range of
cellular locales including the cytosol, mitochondria, actin
cytoskeleton, and endoplasmic reticulum. Previously, a global
SILAC proteomics analysis of amino acid starved MCF7-
eGFP-LC3 cell lysate found that proteins associated with
these locations decreased in abundance to different extents
over a 36 h period.[20] Our observation of these proteins within
acidic organelles after only 1 h of starvation showcases the
sensitivity of our organelle-targeting ABP method. The loss in
the selectivity of DEX-2 for cysteine proteases observed in
autophagic cells is likely due to nucleophilic-residue exposure
resulting from partial degradation of these proteins during
autophagy.
.
[1] N. Li, H. S. Overkleeft, B. I. Florea, Curr. Opin. Chem. Biol.
2012, 16, 227 – 233.
[2] a) A. E. Speers, B. F. Cravatt, Chem. Biol. 2004, 11, 535 – 546;
b) H. C. Hang, J. Loureiro, E. Spooner, A. W. M. van der Vel-
den, Y.-M. Kim, A. M. Pollington, R. Maehr, M. N. Starnbach,
H. L. Ploegh, ACS Chem. Biol. 2006, 1, 713 – 723.
[3] P.-Y. Yang, K. Liu, C. Zhang, G. Y. J. Chen, Y. Shen, M. H. Ngai,
M. J. Lear, S. Q. Yao, Chem. Asian J. 2011, 6, 2762 – 2775.
[4] Y. H. Lee, H. T. Tan, M. C. M. Chung, Proteomics 2010, 10,
3935 – 3956.
[5] a) U. Hillaert, M. Verdoes, B. I. Florea, A. Saragliadis, K. L. L.
Habets, J. Kuiper, S. Van Calenbergh, F. Ossendorp, G. A.
van der Marel, C. Driessen, H. S. Overkleeft, Angew. Chem.
2009, 121, 1657 – 1660; Angew. Chem. Int. Ed. 2009, 48, 1629 –
1632; b) F. Fan, S. Nie, E. B. Dammer, D. M. Duong, D. Pan, L.
Ping, L. Zhai, J. Wu, X. Hong, L. Qin, P. Xu, Y.-H. Zhang, J.
Proteome Res. 2012, 11, 5763 – 5772.
[6] a) D. Greenbaum, A. Baruch, L. Hayrapetian, Z. Darula, A.
Burlingame, K. F. Medzihradszky, M. Bogyo, Mol. Cell. Proteo-
mics 2002, 1, 60 – 68; b) W. W. Kallemeijn, K.-Y. Li, M. D. Witte,
A. R. A. Marques, J. Aten, S. Scheij, J. Jiang, L. I. Willems, T. M.
Voorn-Brouwer, C. P. A. A. van Roomen, R. Ottenhoff, R. G.
Boot, H. van den Elst, M. T. C. Walvoort, B. I. Florea, J. D. C.
Codꢁe, G. A. van der Marel, J. M. F. G. Aerts, H. S. Overkleeft,
Angew. Chem. 2012, 124, 12697 – 12701; Angew. Chem. Int. Ed.
2012, 51, 12529 – 12533.
[7] a) J. Kornhuber, P. Tripal, M. Reichel, C. Mꢂhle, C. Rhein, M.
Muehlbacher, T. W. Groemer, E. Gulbins, Cell. Physiol. Bio-
chem. 2010, 20, 9 – 20; b) Y. Kurishita, T. Kohira, A. Ojida, I.
Hamachi, J. Am. Chem. Soc. 2012, 134, 18779 – 18789.
[8] T. Lꢂbke, P. Lobel, D. E. Sleat, Biochim. Biophys. Acta Mol. Cell
Res. 2009, 1793, 625 – 635.
We observed that enzyme activity increases during
autophagy. Cathepsins B and Z showed a moderate increase
of 1.6-fold (p-value < 0.05). Previous studies show that short-
term starvation and amino acid deprivation induce proteoly-
sis.[13,17b,21] Furthermore, in mouse liver, cathepsin B activity
initially decreases but then increases over 68 h of starva-
tion.[21] These enzyme activity assays were performed on liver
homogenate and may not accurately portray enzyme activity
[9] C. De Duve, T. De Barsy, B. Poole, A. Trouet, P. Tulkens, F.
Van Hoof, Biochem. Pharmacol. 1974, 23, 2495 – 2531.
Angew. Chem. Int. Ed. 2014, 53, 2919 –2922
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
2921

.
Angewandte
Communications
[10] R. G. Anderson, J. R. Falck, J. L. Goldstein, M. S. Brown, Proc.
Natl. Acad. Sci. USA 1984, 81, 4838 – 4842.
Jones, S. Weinman, X.-M. Yin, W.-X. Ding, Autophagy 2011, 7,
188 – 204.
[11] D. Greenbaum, K. F. Medzihradszky, A. Burlingame, M. Bogyo,
Chem. Biol. 2000, 7, 569 – 581.
[12] S. V. Costes, D. Daelemans, E. H. Cho, Z. Dobbin, G. Pavlakis, S.
Lockett, Biophys. J. 2004, 86, 3993 – 4003.
[13] N. Mizushima, A. Yamamoto, M. Matsui, T. Yoshimori, Y.
Ohsumi, Mol. Biol. Cell 2004, 15, 1101 – 1111.
[14] I. Davis, Biochem. Soc. Trans. 2009, 37, 1042 – 1044.
[15] V. Kaminskyy, B. Zhivotovsky, Biochim. Biophys. Acta Proteins
Proteomics 2012, 1824, 44 – 50.
[18] W. A. Dunn, J. Cell Biol. 1990, 110, 1935 – 1945.
[19] J. Dengjel, M. Høyer-Hansen, M. O. Nielsen, T. Eisenberg, L. M.
Harder, S. Schandorff, T. Farkas, T. Kirkegaard, A. C. Becker, S.
Schroeder, K. Vanselow, E. Lundberg, M. M. Nielsen, A. R.
Kristensen, V. Akimov, J. Bunkenborg, F. Madeo, M. Jꢄꢄttelꢄ,
J. S. Andersen, Mol. Cell. Proteomics 2012, 11, M111.014035.
[20] A. R. Kristensen, S. Schandorff, M. Høyer-Hansen, M. O.
Nielsen, M. Jꢄꢄttelꢄ, J. Dengjel, J. S. Andersen, Mol. Cell.
Proteomics 2008, 7, 2419 – 2428.
[16] B. Levine, G. Kroemer, Cell 2008, 132, 27 – 42.
[17] a) D. Muno, N. Sutoh, T. Watanabe, Y. Uchiyama, E. Kominami,
Eur. J. Biochem. 1990, 191, 91 – 98; b) G. Fuertes, J. J. Martꢃn
De Llano, A. Villarroya, A. J. Rivett, E. Knecht, Biochem. J.
2003, 375, 75 – 86; c) H.-M. Ni, A. Bockus, A. L. Wozniak, K.
[21] T. Inubushi, M. Shikiji, K. Endo, H. Kakegawa, Y. Kishino, N.
Katunuma, Biol. Chem. 1996, 377, 539 – 542.
[22] MS/MS spectra for all DEX-2 studies have been uploaded into
PeptideAtlas under dataset identifier PASS00302; http://www.
peptideatlas.org/PASS/PASS00302.
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