Identification and characterization of β-sitosterol target proteins

Article abstract of DOI:10.1016/j.bmcl.2015.03.007

β-Sitosterol is the most abundant plant sterol in the human diet. It is also the major component of several traditional medicines, including saw palmetto and devil's claw. Although β-sitosterol is effective against enlarged prostate in human clinical trials and has anti-cancer and anti-inflammatory activities, the mechanisms of action are poorly understood. Here, we report the identification of two new binding proteins for β-sitosterol that may underlie its beneficial effects.

Full text of DOI:10.1016/j.bmcl.2015.03.007

Bioorganic & Medicinal Chemistry Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification and characterization of b-sitosterol target proteins
a,
⇑
b
a,c,d,
⇑
, Chuo Chen b,
⇑
Brett Lomenick , Heping Shi , Jing Huang
a
Department of Molecular and Medical Pharmacology, 23-231 Center for Health Sciences, 650 Charles E Young Dr. South, University of California Los Angeles, Los Angeles, CA
9
0095, USA
b
Department of Biochemistry, The University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390, USA
Molecular Biology Institute, University of California Los Angeles, Los Angeles, CA 90095, USA
Jonsson Comprehensive Cancer Center, University of California Los Angeles, Los Angeles, CA 90095, USA
c
d
a r t i c l e i n f o
a b s t r a c t
Article history:
b-Sitosterol is the most abundant plant sterol in the human diet. It is also the major component of several
traditional medicines, including saw palmetto and devil’s claw. Although b-sitosterol is effective against
enlarged prostate in human clinical trials and has anti-cancer and anti-inflammatory activities, the
mechanisms of action are poorly understood. Here, we report the identification of two new binding
proteins for b-sitosterol that may underlie its beneficial effects.
Received 11 February 2015
Revised 28 February 2015
Accepted 3 March 2015
Available online xxxx
Ó 2015 Elsevier Ltd. All rights reserved.
Keywords:
b-Sitosterol
1
7b-HSD4
E-Syt1
Target identification
Phytosterol
Phytosterols are a group of steroids produced by plants. They
are structurally and functionally related to cholesterol and
comprise a major component of the human diet. Among them,
which b-sitosterol exerts its many beneficial health effects, we
performed affinity chromatography using biotinylated b-sitosterol
to identify its protein targets.
1,2
b-sitosterol (24-ethylcholesterol) is one of the most abundant diet-
ary phytosterols present in many beans, nuts, and seeds (Fig. 1).
We reasoned that the health promoting effects of b-sitosterol
not observed with cholesterol originate from the existence of
unshared protein targets. b-Sitosterol differs from cholesterol
solely by the presence of an ethyl group at the C-24 position, which
we hypothesized would be an important moiety for the binding of
b-sitosterol specific proteins. Therefore, we prepared affinity
reagents for both compounds by attaching a biotin group to each
through a polyethylene glycol linker (Fig. 1). The C-3 position
was chosen as the attaching point because it is furthest away from
the C-24 position and thus least likely to interfere with proteins
that selectively bind b-sitosterol.
3
–5
It is also an important constituent of saw palmetto, devil’s claw,
stinging nettle and several other natural remedies.6 b-Sitosterol
consumption has been reported to decrease blood cholesterol
levels by preventing its intestinal absorption.9 It also has been
–8
,10
shown to have anti-inflammatory and analgesic properties in
various animal models.1
1–14
Additionally, in both animal models
and human clinical trials, b-sitosterol has demonstrated
a
significant effect on reducing the symptoms of benign prostatic
1
5,16
hyperplasia.
b-Sitosterol intake may also be partially responsi-
ble for the decreased incidence of prostate, colon and breast
cancers among vegetarians and men and women in Asian countries
who consume much larger amounts of b-sitosterol than most
We performed affinity chromatography initially with
lipopolysaccharide (LPS)-treated Raw264.7 macrophage cell
lysates because many of the anti-inflammatory properties of
5
,17
21,22
Westerners.
In support of this hypothesis, b-sitosterol exhibits
b-sitosterol may arise from its effects on macrophages.
The
growth inhibitory and cytotoxic effects against a range of cancer
lysates were first incubated with the biotinylated compounds or
biotin alone as a control at various concentrations for 2 h, followed
by overnight incubation of all with streptavidin agarose resin.
SDS-PAGE and silver staining analysis revealed two bands that
bound specifically to b-sitosterol, a 75-kDa band at 200 nM
7
,18–20
cell lines.
However, the precise molecular mechanisms
underlying its health promoting effects remain largely
uncharacterized. To understand the molecular mechanism(s) by
(
Fig. 2A) and a 120-kDa band at 600 nM (Fig. 2B). MALDI mass
⇑
Corresponding authors. Tel.: +1 310 206 3619 (B.L.); tel.: +1 310 825 4329 (J.H.);
tel.: +1 214 538 7227 (C.C.).
spectrometry analysis of the bands identified them as 17-beta-
hydroxysteroid dehydrogenase 4 (17b-HSD4) and extended
synaptotagmin 1 (E-Syt1).
E-mail addresses: blomenick@mednet.ucla.edu (B. Lomenick), jinghuang@
mednet.ucla.edu (J. Huang), chuo.chen@utsouthwestern.edu (C. Chen).
http://dx.doi.org/10.1016/j.bmcl.2015.03.007
0
960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Lomenick, B.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.03.007

2
B. Lomenick et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
Figure 1. The structures of b-sitosterol, cholesterol, and the affinity reagents used herein.
Figure 2. b-Sitosterol binds to 17b-HSD4 and E-Syt1 in lysates from LPS-treated Raw264.7 mouse macrophages. Affinity chromatography was performed using several
concentrations of biotin (b), biotinylated cholesterol (c), and biotinylated b-sitosterol (s). Silver-staining and mass spectrometry analysis discovered two b-sitosterol specific
binders: (A) 17b-HSD4 at 200 nM and (B) E-Syt1 at 600 nM. Immunoblotting analysis of the affinity chromatography samples validated the binding of (C) 17b-HSD4 at
2
00 nM and (D) E-Syt1 at 600 nM.
To validate these proteins we performed immunoblotting using
likewise validated that E-Syt1 bound specifically to b-sitosterol
(Fig. 2D). At this concentration, 17b-HSD4 appears to be bound
equally well by b-sitosterol and cholesterol, suggesting that the
ethyl group at C-24 in b-sitosterol increases its affinity to
17b-HSD4 but is not necessary for binding.
separate aliquots of the affinity chromatography samples. As
shown in Figure 2C, western blotting with two different 17b-
HSD4 antibodies confirms that it is bound much more strongly
by b-sitosterol than cholesterol. While the full length 17b-HSD4
polypeptide is 79 kDa, a portion of the cellular pool of 17b-HSD4
is proteolytically cleaved into two polypeptides, a 34-kDa N-term-
inal fragment and a 45-kDa C-terminal fragment.23 These two
polypeptides are stable within the cell and are thought to retain
their enzymatic functions, either alone or as homodimers or
Next, we extended our affinity chromatography studies to two
prostate cancer cell lines, PC-3 and DU-145, because b-sitosterol
has been reported to inhibit the growth, migration, and invasion
of prostate cancer cells and is used to treat enlarged prostate.7
As shown in Figure 3, E-Syt1 bound specifically to biotinylated
b-sitosterol at 600 nM, just as in macrophage lysates. In contrast,
17b-HSD4 bound more strongly to biotinylated cholesterol than
biotinylated b-sitosterol in both prostate cancer cell lines (Fig. 3),
which is opposite to what we observed in macrophage lysates.
,15,28
2
4–26
heterodimers.
Although both fragments are present in the
macrophage lysates, only the C-terminal fragment was bound by
b-sitosterol (Fig. 2C). Interestingly, the C-terminal fragment
contains a sterol carrier protein type 2 (SCP-2) domain, which
2
7
may be the site of b-sitosterol and cholesterol binding.
Immunoblotting of the 600 nM affinity chromatography samples
b-Sitosterol has been reported to inhibit 5
a-reductase at micromo-
lar concentrations, and this inhibition has been hypothesized to be
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B. Lomenick et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
3
Figure 3. The binding of b-sitosterol and cholesterol to 17b-HSD4 and E-Syt1 in prostate cancer cell lysates. Affinity chromatography was performed using 200 nM and
00 nM concentrations of biotin (b), biotinylated b-sitosterol (s), and biotinylated cholesterol (c) in (A) PC-3 and (B) DU-145 prostate cancer cell lysates (L). Immunoblotting
6
determined that E-Syt1 binds specifically to b-sitosterol, whereas 17b-HSD4 binds more strongly to cholesterol. S5A1 does not bind either compound at the tested
concentrations.
responsible for its activities in prostate cancer and enlarged
line, it is more likely that the nature of the probes is responsible.
Whereas our sterols were modified at the C-3 position for biotin
labeling, the photoreactive sterol probes were modified on their
alkyl side chains to incorporate alkynes via an ester linkage at
the C-24 position, as well as contain a photoactivatable diazirine
group at the C-6 position. However, since the ethyl group in
b-sitosterol that is absent in cholesterol is at the C-24 position, it
is likely that the addition of an ester linker at C-24 disrupts binding
to 17b-HSD4 in the chemoproteomic experiments. Although the
steroid core of cholesterol was unmodified in the photoreactive
probes with the exception of the diazirine group, which was
hypothesized to maintain binding to most sterol interactors, there
may be a significant number of cholesterol binding proteins that
require the side chain to be unmodified for the interaction to occur.
The primary function of 17b-HSD4 is catalysis of the second and
third steps of peroxisomal b-oxidation, although it can also
2
9
prostate. However, we did not detect binding of 5
S5A1) to biotinylated b-sitosterol or biotinylated cholesterol at
our nanomolar test concentrations (Fig. 3). While this could indi-
cate that the affinity of b-sitosterol for 5 -reductase is lower than
for both 17b-HSD4 and E-Syt1, we cannot rule out the possibility
that the biotin tag interferes with binding to 5 -reductase.
a-reductase
(
a
a
Given the surprising finding that 17b-HSD4 binds to b-sitosterol
more strongly in LPS-treated macrophage lysates but to cholesterol
more strongly in prostate cancer cell lysates, we tested whether
LPS stimulation was responsible for this effect. We observed that
1
7b-HSD4 preferentially bound to b-sitosterol over cholesterol in
macrophage lysates both with and without LPS treatment
Fig. 4). These results suggest that 17b-HSD4 has different affinities
(
to b-sitosterol and cholesterol in different cell lines or cell types,
which could be due to differential splicing, post-translation modi-
fication, protein complex composition, or other factors in each cell
line. Alternatively, there could be a species-specific difference
between the mouse and human 17b-HSD4 homologs, which are
dehydrogenate
D5-androstene-3b,17b-diol and estradiol to the
less potent 17-keto compounds dihydroepiandrosterone and
2
3
estrone. Almost all human tissues possess detectable 17b-HSD
activity, and 17b-HSD4 is thought to be an important housekeep-
ing enzyme responsible for inactivating the most potent estrogen,
8
6% identical.
E-Syt1, along with its homolog E-Syt2, were recently found to
2
7,31,32
bind cholesterol in a chemoproteomic screen using clickable, pho-
17b-estradiol, in all tissues.
While 17b-HSD4 is not known to
3
0
toreactive sterol probes and quantitative mass spectrometry.
play role in inflammation or enlarged prostate, elevated
a
Although we did not detect binding of E-Syt1 to biotinylated
cholesterol, this could be due to the low concentration (200 and
17b-HSD4 expression and activity as well as increased peroxisomal
b-oxidation pathway activity have been found in prostate cancer
tissues compared to normal prostate tissue, and may be indicative
6
1
00 nM) of the probe used in our experiment (compared to
M in the chemoproteomic screen). Moreover, in this chemo-
3
3–35
0
l
of a poor prognosis.
Loss of 17b-HSD4 activity also leads to a
proteomic study, the photoreactive sterol probes were added to
live cells, which were subsequently treated with UV light to cova-
lently crosslink the probes to target proteins. If the interaction
between cholesterol and E-Syt1 is weak or transient it may not
be maintained during the affinity chromatography wash steps,
but it would not be lost by washing the photoreactive probe since
it is covalently bound.
severe d-bifunctional protein deficiency that is usually lethal by
3
2
the age of one. b-Sitosterol may therefore derive its activity
against prostate cancer through modulation of 17b-HSD4 activity.
However, since b-sitosterol does not bind to the N-terminal
domain of 17b-HSD4, it is unlikely to affect its dehydrogenase
activity. It is therefore more likely to affect the activity of the
central dehydratase domain or the C-terminal SCP-2 domain,
whose function is not clear but may be involved in lipid transfer.23
Less is known about the cellular function of E-Syt1. It is local-
ized to the endoplasmic reticulum (ER) where it directly tethers
1
7b-HSD4, on the other hand, was not detected as a cholesterol
binder in the chemoproteomic study despite the much higher con-
centration. Although this could be due to the use of a different cell
Figure 4. Lipopolysaccharide treatment does not affect the binding of 17b-HSD4 or E-Syt1 to b-sitosterol. Affinity chromatography was performed using (A) 200 nM and (B)
00 nM concentrations of biotin (b), biotinylated b-sitosterol (s), and biotinylated cholesterol (c) in lysates from LPS-treated and non-treated Raw264.7 mouse macrophages.
6
Immunoblotting analysis revealed that the binding of 17b-HSD4 and E-Syt1 to cholesterol and b-sitosterol is unchanged by LPS treatment.
Please cite this article in press as: Lomenick, B.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.03.007

4
B. Lomenick et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
the ER membrane and the plasma membrane (PM) in response to
elevated cytosolic calcium.36 E-Syt1 is a two-pass transmembrane
protein that extends into the cytoplasm and binds PI(4,5)P2 in the
this article can be found, in the online version, at http://dx.doi.
org/10.1016/j.bmcl.2015.03.007.
3
6
PM via its C2 domains. Phosphorylation of E-Syt1 by the onco-
genic tyrosine kinase CD74-ROS has been linked to invasiveness
in non-small cell lung cancer, and RNAi knockdown of E-Syt1
attenuated the invasive properties of CD74-ROS expressing cells
References and notes
1
2
3
.
.
.
Ostlund, R. E., Jr. Annu. Rev. Nutr. 2002, 22, 533.
Moreau, R. A.; Whitaker, B. D.; Hicks, K. B. Pro. Lipid Res. 2002, 41, 457.
Weihrauch, J. L.; Gardner, J. M. J. Am. Diet Assoc. 1978, 73, 39.
3
7
in vitro and in vivo. As b-sitosterol has been reported to inhibit
4. Ovesná, Z.; Vachálková, A.; Horváthová, K. Neoplasma 2004, 51, 407.
Awad, A. B.; Fink, C. S. J. Nutr. 2000, 130, 2127.
6. Ludwig-Müller, J.; Georgiev, M.; Bley, T. Process Biochem. 2008, 43, 15.
5.
prostate cancer cell migration and invasion in vitro, it is possible
2
8
this may occur through modulation of E-Syt1 function.
7
.
Scholtysek, C.; Krukiewicz, A. A.; Alonso, J.-L.; Sharma, K. P.; Sharma, P. C.;
Goldmann, W. H. Biochem. Biophys. Res. Commun. 2009, 379, 795.
Nahata, A.; Dixit, V. K. Andrologia 2012, 44, 396.
Vahouny, G. V.; Connor, W. E.; Subramaniam, S.; Lin, D. S.; Gallo, L. L. Am. J. Clin.
Nutr. 1983, 37, 805.
Interestingly, recent evidence suggests that the synaptotagmin-
like mitochondrial-lipid-binding protein (SMP) domain of E-Syt2
functions in lipid transfer between the ER and PM, although bind-
8
9
.
.
3
8
ing of the SMP domain of E-Syt2 to cholesterol was not detected.
10. Ikeda, I.; Tanaka, K.; Sugano, M.; Vahouny, G. V.; Gallo, L. L. J. Lipid Res. 1988, 29,
If cholesterol and b-sitosterol both bind to the SMP domain of
E-Syt1, it is possible that b-sitosterol interferes with the lipid
transfer activity of E-Syt1, and perhaps the other E-Syt proteins
as well. Hypercholesterolemia is a risk factor for estrogen recep-
tor-positive breast cancer, the pathology of which is mediated
via its conversion to the estrogen receptor and liver X receptor
ligand 27-hydroxycholesterol.39 It will be interesting to determine
if b-sitosterol inhibits the transfer or conversion of cholesterol and
its metabolites in pre-malignant and transformed breast cancer
cells.
1573.
1
1
1. Gupta, M. B.; Nath, R.; Srivastava, N.; Shanker, K.; Kishor, K.; Bhargava, K. P.
Planta Med. 1980, 39, 157.
2. Villaseñor, I. M.; Angelada, J.; Canlas, A. P.; Echegoyen, D. Phytother. Res. 2002,
16, 417.
1
1
1
3. Mahajan, S. G.; Mehta, A. A. Eur. J. Pharmacol. 2011, 650, 458.
4. Lee, I.-A.; Kim, E.-J.; Kim, D.-H. Planta Med. 2012, 78, 896.
5. Berges, R. R.; Windeler, J.; Trampisch, H. J.; Senge, T. Lancet 1995, 345, 1529.
16. Klippel, K. F.; Hiltl, D. M.; Schipp, B. Br. J. Urol. 1997, 80, 427.
1
1
7. Woyengo, T. A.; Ramprasath, V. R.; Jones, P. J. Eur. J. Clin. Nutr. 2009, 63, 813.
8. Moon, D. O.; Lee, K. J.; Choi, Y. H.; Kim, G. Y. Int. Immunopharmacol. 2007, 7,
1044.
19. Park, C.; Moon, D. O.; Rhu, C. H.; Choi, B. T.; Lee, W. H.; Kim, G. Y.; Choi, Y. H.
Biol. Pharm. Bull. 2007, 30, 1317.
In summary, we report the identification of two new binding
proteins for b-sitosterol that has been shown to be effective against
enlarged prostate in human clinical trials. b-Sitosterol also has
anti-cancer and anti-inflammatory activities in cell culture
20. Park, C.; Moon, D. O.; Ryu, C. H.; Choi, B. T.; Lee, W. H.; Kim, G. Y.; Choi, Y. H.
Acta Pharmacol. Sin. 2008, 29, 341.
21. Ding, Y.; Nguyen, H. T.; Kim, S. I.; Kim, H. W.; Kim, Y. H. Bioorg. Med. Chem. Lett.
009, 19, 3607.
2
2
2
2. Alappat, L.; Valerio, M.; Awad, A. B. Int. Immunopharmacol. 2010, 10, 1390.
3. Huyghe, S.; Mannaerts, G. P.; Baes, M.; Van Veldhoven, P. P. Biochim. Biophys.
Acta 2006, 1761, 973.
(
including prostate, breast, colon, leukemia, T-cells, and macro-
phages). Previously, only one enzyme (5 -reductase) was known
to be inhibited by b-sitosterol. It remains unclear if inhibition of
-reductase is responsible for the effects of b-sitosterol on benign
a
24. Adamski, J.; Leenders, F.; Carstensen, J. F.; Kaufmann, M.; Markus, M. M.;
Husen, B.; Tesdorpf, J. G.; Seedorf, U.; de Launoit, Y.; Jakob, F. Steroids 1997, 62,
5
a
1
59.
5. de Launoit, Y.; Adamski, J. J. Mol. Endocrinol. 1999, 22, 227.
26. Jiang, L. L.; Miyazawa, S.; Souri, M.; Hashimoto, T. J. Biochem. 1997, 121, 364.
prostatic hyperplasia and prostate cancer, and modulation of
additional targets may be essential. Moreover, inhibition of
5a-reductase is unlikely to play a role in the anti-inflammatory
2
27. Adamski, J.; Normand, T.; Leenders, F.; Monté, D.; Begue, A.; Stéhelin, D.;
Jungblut, P. W.; de Launoit, Y. Biochem. J. 1995, 311, 437.
activity of b-sitosterol or in its effects on other cancers. Using an
affinity chromatography approach, we provide evidence that
b-sitosterol directly binds to 17b-HSD4 and E-Syt1. These novel
targets may be responsible for many of the beneficial effects of
b-sitosterol. The two biotinylated affinity probes may also serve
as useful tools for further comprehensive analysis of the sterol
binding subset of the proteome.
2
8. Awad, A. B.; Fink, C. S.; Williams, H.; Kim, U. Eur. J. Cancer Prev. 2001, 10, 507.
29. Cabeza, M.; Bratoeff, E.; Heuze, I.; Ramírez, E.; Sánchez, M.; Flores, E. Proc. West.
Pharmacol. Soc. 2003, 46, 153.
30. Hulce, J. J.; Cognetta, A. B.; Niphakis, M. J.; Tully, S. E.; Cravatt, B. F. Nat. Methods
2013, 10, 259.
31. Martel, C.; Rhéaume, E.; Takahashi, M.; Trudel, C.; Couët, J.; Luu-The, V.;
Simard, J.; Labrie, F. Steroid Biochem. Mol. Biol. 1992, 41, 597.
3
3
2. Möller, G.; Adamski, J. Mol. Cell. Endocrinol. 2006, 248, 47.
3. Zha, S.; Ferdinandusse, S.; Denis, S.; Wanders, R. J.; Ewing, C. M.; Luo, J.; De
Marzo, A. M.; Isaacs, W. B. Cancer Res. 2003, 63, 7365.
3
3
4. Zha, S.; Ferdinandusse, S.; Hicks, J. L.; Denis, S.; Dunn, T. A.; Wanders, R. J.; Luo,
J.; De Marzo, A. M.; Isaacs, W. B. Prostate 2005, 63, 316.
5. Rasiah, K. K.; Gardiner-Garden, M.; Padilla, E. J.; Möller, G.; Kench, J. G.; Alles,
M. C.; Eggleton, S. A.; Stricker, P. D.; Adamski, J.; Sutherland, R. L.; Henshall, S.
M.; Hayes, V. M. Mol. Cell. Endocrinol. 2009, 301, 89.
Acknowledgments
Support for this research is provided by the NIH National Center
for Complementary and Alternative Medicine (R01-AT006889 to
C.C. and J.H.). B.L. is supported by the NIH-UCLA Tumor
Immunology Training Program (T32-CA009120). We thank Yi-Pei
Chen and Tremylla Johnson for technical assistance.
3
6. Giordano, F.; Saheki, Y.; Idevall-Hagren, O.; Colombo, S. F.; Pirruccello, M.;
Milosevic, I.; Gracheva, E. O.; Bagriantsev, S. N.; Borgese, N.; De Camilli, P. Cell
2013, 153, 1494.
37. Jun, H. J.; Johnson, H.; Bronson, R. T.; de Feraudy, S.; White, F.; Charest, A.
Cancer Res. 2012, 72, 3764.
3
3
8. Schauder, C. M.; Wu, X.; Saheki, Y.; Narayanaswamy, P.; Torta, F.; Wenk, M. R.;
De Camilli, P.; Reinisch, K. M. Nature 2014, 510, 552.
9. Nelson, E. R.; Wardell, S. E.; Jasper, J. S.; Park, S.; Suchindran, S.; Howe, M. K.;
Carver, N. J.; Pillai, R. V.; Sullivan, P. M.; Sondhi, V.; Umetani, M.; Geradts, J.;
McDonnell, D. P. Science 2013, 342, 1094.
Supplementary data
Supplementary data (methods for biological and biochemical
assays, MALDI mass spectrometry data, procedures for chemical
synthesis, and compound characterization data) associated with
Please cite this article in press as: Lomenick, B.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.03.007