Discovery of small peptides derived from embryonic lethal abnormal vision proteins structure showing RNA-stabilizing properties

Article abstract of DOI:10.1021/jm900741e

Four peptides corresponding to highly conserved motives within the first two RNA recognition motif-type domains of ELAV proteins were prepared, and their effect on the stability of NOVA-1 and VEGF ELAV-target mRNAs was evaluated. Biological results show t

Full text of DOI:10.1021/jm900741e

J. Med. Chem. 2009, 52, 5017–5019 5017
DOI: 10.1021/jm900741e
in their cytoplasmic stability and rate of translation occurs.1,2 In
vertebrates, HuB, HuC, and HuD are neuron-specific
(nELAV) while HuR is ubiquitously expressed.3
Discovery of Small Peptides Derived from
Embryonic Lethal Abnormal Vision Proteins
Structure Showing RNA-Stabilizing Properties
Biologically, ELAV are pleiotropic proteins that are im-
portant in various contexts, since they can affect the fate of
some mRNAs whose corresponding proteins are fundamental
for key cellular functions, such as maintenance of cellular
homeostasis, growth and development, survival/apoptosis,
and stress response.4-6 In particular, within brain physiology
nELAV positively control (spatiotemporally) the fate of target
mRNAs whose products are necessary for neuronal differen-
tiation and plasticity, thus making nELAV major determi-
nants of brain development and memory processes.7-9
Consequently, a deficit in the nELAV cascade may play a
key role in pathologies associated with memory disturbances
as indicated by recent findings on Alzheimer’s dementia,10
strongly suggesting the importance of post-transcriptional
regulatory mechanisms on gene expression in the etiology of
the disease.
Daniela Rossi,† Marialaura Amadio,‡
Anna Carnevale Baraglia,† Ornella Azzolina,†
Antonia Ratti,§ Stefano Govoni,‡ Alessia Pascale,*,‡ and
Simona Collina*,†
†Department of Pharmaceutical Chemistry, University of Pavia,
Viale Taramelli 12, I-27100 Pavia, Italy, ‡Department of
Experimental and Applied Pharmacology, University of Pavia,
Viale Taramelli 14, I-27100 Pavia, Italy, and §Department of
Neurological Sciences, “Dino Ferrari” Centre, University of Milan
Medical School, IRCCS Istituto Auxologico Italiano, Via Zucchi 18,
I-20095 Cusano, Milan, Italy
Received May 29, 2009
Abstract: Four peptides corresponding to highly conserved motives
within the first two RNA recognition motif-type domains of ELAV
proteins were prepared, and their effect on the stability of NOVA-1
and VEGF ELAV-target mRNAs was evaluated. Biological
results show that in the presence of phorbol 12-myristate 13-acetate
(a PKC activator triggering the ELAV pathway), an equimolar
mixture of peptides induces a statistically significant stabilization
of the selected transcripts, suggesting a synergic effect of the two
stimuli.
On the basis of these observations, we addressed our efforts
to the discovery of ELAV-mimicking molecules that can be
useful for a basic research aimed at better understanding of
mRNA-protein interactions and can constitute the first step
toward a highly innovative pharmacological approach to
increase or recover the expression of ELAV-target mRNAs
that are defective in some pathological conditions. To date,
the ELAV/mRNA complex remains completely unexplored
from a medicinal chemistry point of view. Currently in the
literature there is no evidence of molecules that may affect the
fate of mRNAs and thus the amount of the corresponding
proteins. Therefore, to find ELAV-mimicking molecules, we
followed a rational design approach based on ELAV proteins
structure analysis and on the resolved 3D-structure of HuD in
complex with a target transcript.
The four vertebrate ELAV proteins are characterized by a
high degree of sequence homology (70-85%). They are about
40 kDa in size and contain three ∼90-amino-acid-long RNA
recognitionmotif-type (RRM) domains. The first two RRMs,
positioned atthe N-terminal, areseparated fromthethirdone,
at the C-terminus, by a hinge segment.3,11 In vitro binding
studies indicate that the first and second RRMs are primarily
implicated in the interaction of ELAV RBPs with the ARE
sequences present in the target mRNA.12,13 Structural inves-
tigation of the first two RRMs bound to ARE shows that they
form a cleft with their β-sheet-containing surfaces in which the
mRNA element is inserted. Nothing is known about the
binding mode of the third RRM to mRNA. This RRM seems
to have affinity for the poly(A) tail of the messenger, thus
contributing to the stability of the ELAV/mRNA complex.4
Moreover, sequence alignment studies of the different ELAV
proteins clearly indicate that the first two RRM domains each
containtwo highlyconserved sequences ofeight and six amino
acids, named RNP1 and RNP2, respectively. Additionally,
X-ray crystallography reveals that the four RNPs (RNP1 and
RNP2 sequences of both RRM1 and RRM2 domains) are
directly involved in the mRNA binding.14
Evidence in the literature indicates an increasing interest in
post-transcriptional regulatory mechanisms because of their
fine, localized effects on gene expression. Within this context,
modulation of mRNA decay appears to be an efficient post-
transcriptional way of controlling gene expression because
small changes in mRNA half-life can drastically alter the
amount of the corresponding protein. Among the different
players, the RNA-binding proteins (RBPsa) can affect the
processing of selected transcripts, their transport and subcel-
lular localization in the cytoplasm, and their stability and
translatability. Among the best characterized RBPs are the
ELAV (embryonic lethal abnormal vision) proteins that pre-
ferentially interact with adenine and uridine-rich elements
(AREs) present in the 30-untranslated region of a subset of
mRNAs. ARE-containing mRNAs are prone to a rapid
degradation, but following the binding of ELAV, an increase
*To whom correspondence should be addressed. For A.P.: phone,
þ39-0382987230; fax, þ39-0382987405; e-mail, alessia.pascale@unipv.
it. For S.C.: phone, þ39-0382987379; fax, þ39-0382422975; e-mail,
simona.collina@unipv.it.
a Abbreviations: ARE, adenine and uridine-rich elements; DRB, 5,6-
dichlorobenzamidazole riboside; ELAV, embryonic lethal abnormal
vision; Fmoc, 9-fluorenylmethyloxycarbonyl; HBTU, 2-(1H-benzotria-
zol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; HOBt, N-
hydroxybenzotriazole; HPLC, high performance liquid chromatogra-
phy; LC-MS, liquid chromatography-mass spectrometry; nELAV,
neuron-specific embryonic lethal abnormal vision proteins; NOVA-1,
neuro-oncological ventral antigen-1; PKC, protein kinase C; P1-4
,
equimolar mixture of peptides 1-4 (1 μM each peptide); PMA, phorbol
12-myristate 13-acetate; RBPs, RNA-binding proteins; RNP, ribonu-
cleoproteic; RRM, RNA recognition motif type; RT-PCR, real time
polymerase chain reaction; SAR, structure-activity relationship;
TBTU, O-(benzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium tetra-
fluoroborate; VEGF, vascular endothelial growth factor.
On the basis of these observations and considering that
peptides are adequate molecules for mimicking protein bind-
ing sites,15 we focused our attention on the preparation and
r
2009 American Chemical Society
Published on Web 07/31/2009
pubs.acs.org/jmc

5018 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16
Rossi et al.
Table 1. Peptide Sequences and Their Correspondence in ELAV Struc-
ture
peptide
sequence
ELAV refa
1
2
3
4
LGYGFVNY
LIVNYL
RRM1_RNP1
RRM1_RNP2
RRM2_RNP1
RRM2_RNP2
RGVGFIRF
LYVSGL
a Correspondence in ELAV structure.
Table 2. Peptide Crude Purities
crude purity,a %
Figure 1. Evaluation of NOVA-1 and VEGF mRNAs decay by
real-time quantitative PCR in SH-SY5Y human neuroblastoma
cells in an 8 h time interval. RPL6 was used as reference mRNA for
NOVA-1 and VEGF normalization, remaining substantially stable
in the 8 h time frame.
peptide
method 1
method 2
1
2
3
4
75.1
66.4
43.6
72.4
90.1
89.3
85.3
90.3
a Determined by analytical reverse-phase HPLC using a Phenomenex
Jupiter C18 column with gradient elution (water:acetonitrile:trifluor-
oacetic acid, see Supporting Information); UV detection at 230 nm.
biological evaluation of four peptides corresponding to
ELAV RNP1 and RNP2 sequences of RRM1 and RRM2
domains (peptides 1-4, Table 1).
Peptides were synthesized according to the solid phase
approach using the Fmoc-based methodology. A parallel
solid-phase procedure was first experimented in an automated
parallel organic synthesizer (Vantage-aapptec) at room tem-
perature, using HBTU and HOBt as the coupling reagents
(method 1; see Supporting Information). After cleavage and
side chain deprotection (trifluoroacetic acid/dichloro-
methane, 95:5 v/v), peptides 1-4 were obtained as main
reaction products (reverse-phase LC-MS analysis; see
Supporting Information) with satisfactory crude purities
(>65%, Table2), with the only exception of 3 (43.6%,
Table 2). A more convenient synthetic procedure, combining
solid-phase peptide synthesis with microwave irradiation, was
then developed using the Liberty microwave-assisted auto-
matic peptide synthesizer (CEM, Matthews, NC) and success-
fully applied to the preparation of 1-4 (method 2; see
Supporting Information). Coupling reactions were performed
under microwave irradiation (80 °C, 25 W, 300 s), using
TBTU. Cleavage and side chain deprotection were then
carried out (trifluoroacetic acid/water/triisopropylsilane,
95:2.5:2.5 v/v/v, 40 °C, 15 W, 25 min), providing the desired
peptides with purities higher than 85.0% (Table 2). Interest-
ingly, a noticeable improvement of the peptide crude purities
was observed when our optimized microwave-assisted proto-
col was applied instead of the conventional parallel one. As
clearly reported in Table 2, this effect was particularly pro-
nounced for peptide 3.
Crudes 1-4 were finally purified by preparative reverse-
phase LC-MS, using a Waters Simmetry C18 column (Waters
autopurification system; see Supporting Information). Frac-
tions of adequate HPLC homogeneity and with the expected
mass were combined and lyophilized, obtaining 1-4 with a
purity higher than 95.0%, as evidenced by the reverse-phase
HPLC analysis (see Supporting Information).
Moving from chemistry to biological investigation, our
strategy consisted of evaluating the capability of peptides
1-4, corresponding to the highly conserved sequences within
the first two ELAV RRM domains, to affect the mRNA
stability without the remaining portion of the protein.
According to our approach, we performed the biological
Figure 2. Effect of P1-4 on VEGF and NOVA-1 mRNA stability:
ratios between percentages (PMA/CON and (PMA þ P1-4)/P1-4
)
of remaining NOVA-1 (A) and VEGF (B) mRNAs in SH-SY5Y
cells after 2 h. The treatments were performed in the presence of
transcriptional blocking: (//) p < 0.005, two-tailed t test, n = 5,
CON = control.
investigation by assaying 1-4 in mixture and not individually,
even if this is not the most commonly used procedure. In
detail, we evaluated the effect of the equimolar (1 μM) mixture
of 1-4 (P1-4) on the stability of two ARE-bearing ELAV
target mRNAs, NOVA-1 (neuro-oncological ventral antigen-
1)9 and VEGF (vascular endothelial growth factor).16
NOVA-1 is a neuron-specific splicing factor that regulates
the alternative processing of a set of transcripts essential for
proper neural development and for synaptic plasticity in the
nervous system.9,17 Indeed, the lack of expression of Nova-1 is
responsible for severe defects in the development of the motor
system in the null mice.18 VEGF is a protein with well-
documented effects on blood vessel formation; moreover, it
has also been related to repairing processes after brain in-
jury.19 Indeed, the deletion of a single Vegf allele causes
embryonic lethality.20
Preliminary kinetic studies aimed at evaluating NOVA-1
and VEGF mRNAs decay (within 8 h in conditions of
transcriptional arrest) led us to set 2 h as the optimal experi-
mental time for performing mRNA stability experiments,
since the mRNA levels of both transcripts are easily detectable
at this time (Figure 1).
The effect of P1-4 on NOVA-1 and VEGF mRNAs stabi-
lity was then evaluated in vitro on SH-SY5Y human neuro-
blastoma cells by quantitative real time PCR (RT-PCR),

Letter
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16 5019
after transcriptionalblock, withor without PMA (phorbol12-
myristate 13-acetate) stimulation (100 nM for 15 min) (see
Supporting Information). PMA is an activator of protein
kinase C able to trigger the downstream phosphorylation/
activation of ELAV proteins.21 Regarding NOVA-1 mRNA,
PMA alone does not produce any change in its stability with
respect to the control (Figure 2A), in accordance with pre-
viously published data.9 Instead, the increase in VEGF
mRNA stability after PMA alone (Figure 2B) is in line with
the higher levels of VEGF protein found in the same experi-
mental conditions in another cell line.16 Results reported in
Figure 2 clearly indicate that the concomitant cell treatment
with P1-4 and PMA gives rise to a statistically significant
stabilization of both ELAV-target transcripts with respect to
PMA alone, while P1-4 without PMA does not show any
detectable mRNA stabilizing effect. This result is of particular
relevance in the case of NOVA-1: only the copresence of the
P1-4 and PMA leads to significant stabilization of its mRNA,
with the PMA alone proving ineffective. In short, results
strongly suggest a synergic effect of P1-4 and PMA stimuli
onmRNAstability, accordingtoourmodel inwhich P1-4 acts
in concert with the PMA-activated ELAV proteins physiolo-
gically present in the cells, through the binding to ARE
sequences and the following stabilization of the target
mRNAs. However, further investigation is needed to better
clarify the mechanism of action of these peptides.
To conclude, we have reported the synthesis and the
biological investigation of four peptides as prototypes of the
first class of ELAV-mimicking molecules. The surprising
discovery made in this study is that the mixture of peptides
1-4 induces a statistically significant stabilization of the two
considered ELAV-target transcripts with respect to PMA
alone. Actually, this is the first time that ELAV mimicking
properties have been clearly indicated for nonphysiological
molecules. Notably, given the pleiotropic action of ELAV,7
the availability of specific molecules able to modulate the fate
of a subset of mRNAs may have relevance in many physio-
logic and pathologic fields.
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^
ꢀ
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Our current efforts are directed toward a deeper under-
standing of the mRNA-stabilizing activity of 1-4 by testing
them individually or in different compositions. Successively,
we intend to address our attention to peptide structure-
activity relationship (SAR) studies and to peptide bioactive
conformation identification followed by design and synthesis
of 1-4 analogues, with the final aim of obtaining new
molecules with improved ELAV mimicking properties.
(15) Eichler, J. Peptides as protein binding site mimetics. Curr. Opin.
Chem. Biol. 2008, 12, 1–7.
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Neurosci. Res. 2002, 68, 121–126.
Acknowledgment. We thank CEM Corporation for the
use of Liberty instrument and Italfarmaco SpA (Cinisello
Balsamo, Milano) for assistance with peptide synthesis and
analysis.
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neuron-specific alternative splicing and is essential for neuronal
viability. Neuron 2000, 25, 359–371.
(19) Krum, J. M.; Mani, N.; Rosenstein, J. M. Roles of the endogenous
VEGF receptors flt-1 and flk-1 in astroglial and vascular remodel-
ing after brain injury. Exp. Neurol. 2008, 212, 108–117.
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O’Shea, K. S.; Powell-Braxton, L.; Hillan, K. J.; Moore, M. W.
Heterozygous embryonic lethality induced by targeted inactivation
of the VEGF gene. Nature 1996, 380, 439–442.
Supporting Information Available: Details of syntheses, puri-
fication, characterization, and biological evaluation of peptides
1-4; HPLC chromatograms. This material is available free of
charge via the Internet at http://pubs.acs.org.
(21) Pascale, A.; Amadio, M.; Scapagnini, G.; Lanni, C.; Racchi, M;
Provenzani, A.; Govoni, S.; Alkon, D. L.; Quattrone, A. Neuronal
ELAV proteins enhance mRNA stability by a PKCalpha-depen-
dent pathway. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 12065–
12070.
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