This enzyme, TcrPDEC, is a member of the class I PDEs, as determined from the presence of a characteristic signature sequence and from the conservation of a number of functionally import
Trang 1phosphodiesterase from Trypanosoma cruzi
Stefan Kunz, Michael Oberholzer and Thomas Seebeck
Institute of Cell Biology, University of Bern, Bern, Switzerland
The cell biology of Trypanosoma cruzi, the causative
agent of South American Chagas’ disease, has been
extensively studied Surprisingly, still very little is
known about the role of cyclic nucleotide signaling in
this organism [1] A number of earlier studies have
indicated a role of cAMP in differentiation [2,3], and
the existence of a nitric oxide regulated guanylyl
cyclase has been suggested [4] In T cruzi
epimasti-gotes, a cAMP-regulated transcript has been identified
that can be induced by elevated cAMP levels [5] The
corresponding gene, TC26, was later found to code for
an RNaseH and to be localized on a large family of
repetitive genetic elements More recently, the T cruzi
genome was shown to code for several adenylyl
cyclas-es, all predicted to be similarly organized, consisting of
a large N-terminal, presumably extracellular region, which is followed by a single transmembrane helix and
a C-terminal catalytic domain [6] The structure of these cyclases is entirely different from that of their mammalian counterparts, but closely similar to that of the cyclases characterized in Leishmania donovani [7] and in African trypanosomes [8–10] One of these adenylyl cyclases, TczAC, was found to interact with a paraflagellar rod protein, and is most likely located in the flagellum [11]
A cAMP-specific phosphodiesterase (PDE) activity has been demonstrated in T cruzi by various laborat-ories [12,13] Recently, the first cyclic-nucleotide-specific PDE from T cruzi has been identified and characterized at the molecular level [14] This enzyme,
Keywords:
Chagas’ disease; cyclic nucleotides; FYVE
domain; kinetoplastids; phosphodiesterase
Correspondence
T Seebeck, Institute of Cell Biology,
Baltzerstrasse 4, CH-3012 BERN,
Switzerland
Fax: +41 31 6314684
Tel: +41 31 6314649
E-mail: thomas.seebeck@izb.unibe.ch
Website: http://www.izb.unibe.ch
Nucleotide sequence data have been
sub-mitted to the DDBJ ⁄ EMBL ⁄ GenBank
data-bases under the accession numbers
AJ889575 and AJ889576 for TcrPDEC
alle-les 1 and 2, respectively.
(Received 26 August 2005, revised 20
Octo-ber 2005, accepted 27 OctoOcto-ber 2005)
doi:10.1111/j.1742-4658.2005.05039.x
Cyclic-nucleotide-specific phosphodiesterases (PDEs) are key players in the intracellular signaling pathways of the important human pathogen Trypano-soma cruzi We report herein the identification of an unusual PDE from this protozoal organism This enzyme, TcrPDEC, is a member of the class
I PDEs, as determined from the presence of a characteristic signature sequence and from the conservation of a number of functionally important amino acid residues within its catalytic domain Class I PDEs include a large number of PDEs from eukaryotes, among them all 11 human PDE families Unusually for an enzyme of this class, TcrPDEC contains a FYVE-type domain in its N-terminal region, followed by two closely spaced coiled-coil domains Its catalytic domain is located in the middle of the polypeptide chain, whereas all other class I enzymes contain their cata-lytic domains in their C-terminal parts TcrPDEC can complement a PDE-deficient yeast strain Unexpectedly for a kinetoplastid PDE, TcrPDEC is a dual-specificity PDE that accepts both cAMP and cGMP as its substrates
Abbreviations
DMSO, dimethyl sulfoxide; EHNA, erythro-9-(2-hydroxy-3-nonyl)adenosin; FYVE, domain containing Fab1p, YOTB, Vac1p and EEA1PDE; GST, glutathione-S-transferase; IBMX, isobutyl methyl xanthine; LmPDEC, phosphodiesterase from Leishmania major; PtdIns(3)P, phosphatidyl inositol-3-phosphate; TbPDEC, phosphodiesterase from Trypanosoma brucei; TcrPDEC, phosphodiesterase from Trypanosoma cruzi.
Trang 2TcPDE1 is entirely cAMP-specific, and it is located
along the flagellum In terms of its amino acid
sequence, TcPDE1 is a close homologue of the
Trypano-soma bruceiPDEs TbPDE2B [15] and TbPDE2C [16],
as well as of LmPDEB1 and LmPDEB2 of Leishmania
major (Johner et al., unpublished results) All of these
enzymes belong to the class I PDEs [17] TcPDE1
con-tains two GAF domains [18,19] in its N-terminal
moi-ety, and a C-terminal catalytic domain TcPDE1, as all
its other kinetoplastid homologues, is highly
cAMP-selective
This study reports the identification and
characteri-zation of a novel and rather unusual PDE from T cruzi
According to the recently proposed unifying
nomen-clature for kinetoplastid PDEs [20], this enzyme was
designated as TcrPDEC Based on the amino acid
sequence of its catalytic domain, TcrPDEC
unambigu-ously belongs to the class I PDEs However, it is a
rather unusual PDE in several respects: (a) unlike all
other class I PDEs, its catalytic domain is localized in
the middle of the polypeptide chain, and not at its
C-terminus; (b) the N-terminal region of TcrPDEC
contains a FYVE-type domain [21,22], a functional
domain that has not been found in any PDE so far;
and (c) TcrPDEC is the first dual-substrate PDE, with
similar Kmvalues for cAMP and cGMP, that has been
identified in kinetoplastids
Results
Identification of TcrPDEC
When the T cruzi database (http://www.genedb.org/
genedb/tcruzi) was screened for putative PDEs, a gene
was identified that codes for a rather unusual PDE,
TcrPDEC (temporary gene identification number
Tc00.1047053506697.20) The open reading frame of
TcrPDEC was amplified from genomic DNA, and
sev-eral PCR products were sequenced This analysis
revealed the presence of two distinct alleles which
dif-fer by 62 bp (out of the 2775 bp of the entire open
reading frame; 2.2% sequence divergence) These single
nucleotide polymorphisms translate into 38 amino acid
changes (4.1% amino acid substitutions; 21 conserved,
17 nonconserved) Only six of these substitutions occur
in the catalytic domain of the enzyme, and none of
them affects a residue that is crucial for function (see
below) Southern blot analysis of T cruzi genomic
DNA by hybridization with the complete open reading
frame of TcrPDEC results in restriction enzyme
pat-terns that are compatible with the nucleotide sequence
of TcrPDEC, demonstrating that it represents a single
copy gene (Fig 1) EcoRI, PstI and EcoRV cut once
within the open reading frame, BamHI does not cut, and HindIII cuts three times, resulting in two frag-ments too small to be detected by hybridization and one fragment of two kilobases The hybridization of SalI-digested DNA confirmed the polymorphism of one SalI site detected by the sequence analysis of the two alleles The establishment of TcrPDEC as a single-copy gene is not entirely trivial, as large parts of the
T cruzigenome have undergone a duplication [23]
Functional domains of TcrPDEC The open reading frame of TcrPDEC codes for a pro-tein of 924 amino acids (calculated relative molecular mass of 103 169, calculated pI¼ 5.91) with several functional domains (Fig 2A) The N-terminus (P10–
G73) contains a FYVE-type domain, an acronym composed of the designations of the first four repre-sentatives Fab1p, YOTB, Vac1p and EEA1 [21], that is followed by two closely spaced coiled-coil regions (D144–D179 and K207–E264) FYVE-type domains are zinc-finger-like structures, which are currently divided
10 8 6 5 4
3
2 1.5
1
0.5
EcoRI SalI EcoRV BamHI PstI HindIII
kb
Fig 1 TcrPDEC is a single copy gene Genomic DNA of T cruzi hybridized with a probe representing the entire open reading frame
of TcrPDEC.
Trang 3into two classes: The classical FYVE domains
(exam-ples: (HsEEA1, accession number Q15075; DmHRS,
accession number Q960· 8; and ScVps27p, accession
number P40343) share three consensus motifs (WXXD,
R + HHCR and RVC), and they bind specifically
to
membrane-embedded-phosphatidyl-inositol-3-phos-phate [PtdIns(3)P] FYVE-variant domains such as
HsDFCP1 and AtPRAF1 lack some of the conserved
residues (Fig 2B), but still bind PtdIns(3)P, albeit with
lower affinity The FYVE-related domains (e.g
rabphi-lin 3 A (P47709) or human Rim1 (Q86UR5) exhibit
a still higher sequence divergence in the consensus
region Their function is still undetermined The
align-ment of the FYVE-type domain of TcrPDEC places it
close to the FYVE-variant domains All eight cysteine
residues predicted to be involved in Zn2+-binding are
fully conserved (Fig 2C), as are the two predicted
heli-cal regions The two hydrophobic amino acids that are
inserted in the membrane upon PtdIns(3)P binding
(L185 and L186 in ScVps27p [22]); are represented by
L30 and F31 of TcrPDEC When matched with the
WxxD R + HHCR RVC motif of FYVE domains, the sequence of TcrPDEC exhibits several alterations
A glutamate residue at position four of the first block
is substituted by aspartate, arginine at the beginning of the second block is replaced by an alanine, the two adjacent histidine residues are replaced by serine and glutamine, the subsequent arginine is replaced by pro-line, and finally the arginine of the third block is replaced by a lysine The consequence of these replace-ments is a decrease of the overall net charge of the motive from +4 to +1 The effect of these changes
on a putative membrane binding of the FYVE-type domain of TcrPDEC remains to be explored, but they render the TcrPDEC domain unlikely to bind to PtdIns(3)P This prediction is confirmed by the obser-vation that the recombinant FYVE domain of TcrPDEC does not bind to PtdIns(3)P, nor to PtdIns(3,4)P2, PtdIns(4,5)P2, PtdIns(3,4,5)P3, phos-phatidic acid, phosphatidyl choline, phosphatidyl ser-ine or phosphatidyl inositol in a dot-spot assay [24,25] (data not shown)
FYVE DmHrs
PHD HsKAP-1
FYVE-related HsRIM1
FYVE-related RnRPH3A
TcPDEC
FYVE-variant AtPRAF1
FYVE-variant HsDCFP1
FYVE ScVps27p
FYVE HsEEA1
C
Fig 2 The FYVE-type domain of TcrPDEC (A) Functional organization of TcrPDEC: 1, FYVE-type domain; 2 and 3, coiled-coil regions; 4, cata-lytic domain (B) Dendrogram of FYVE domains: TcrPDEC; HsEEA1, human early endosome antigen 1 (accession number Q15075); DmHrs, Drosophila Hrs (Q960 · 8); ScVps27p, S cerevisiae vacuolar sorting protein (P40343); HsDFCP1, human double FYVE-containing protein 1 (Q9HBF4); AtPRAF1, Arabidopsis PRAF1 (Q947D2); RnRPH3A, rat rabphilin-3 A (P47709); HsRIM1, human Rim1 (Q86UR5) (C) Alignment of FYVE and FYVE-related domains Grey boxes: the conserved Zn 2+ coordinating cysteine residues The FYVE domain signature motifs WxxD,
R + HHCRxCG and RVC are given in bold, underlined letters Box 1: turret loop [41]; box 2: a-helix found in the structures of HsEEA1, DmHrs, ScVps27p and RnRabphilin-3 A Horizontal box: putative dimer interface of EEA1 [42].
Trang 4Downstream of the FYVE domain, TcrPDEC is
pre-dicted to contain two closely spaced coiled-coil regions
(S150–L174and K207–D264) These might serve to
dimer-ize the FYVE domains in a way similar to the
struc-ture that was determined for EEA1 [26]
To explore if these regions are indeed essential for
stabilizing the FYVE domain in the dimeric state, the
FYVE domain was expressed either alone (amino acids
1–74 of TcrPDEC), or in conjunction with the
coiled-coil region (amino acids 1–272) Gel filtration analysis
demonstrated that already the FYVE domain alone
migrates as a stable dimer (calculated molecular mass
8.2 kDa; apparent molecular mass upon gel filtration:
16.2 kDa) (Fig 3A) The construct containing the two
coiled-coil regions in addition to the FYVE domain (calculated molecular mass 30.5 kDa) eluted with an apparent mass of 199.7 kDa, indicating the formation
of a higher order complex (Fig 3B)
In TcrPDEC, the catalytic domain is located in the middle of the polypeptide chain of 924 amino acids (T291–S657; Fig 2A) This is very unusual for a class I PDE, as all other members of this PDE class contain the catalytic domain in their C-terminal portions Nev-ertheless, the catalytic domain of TcrPDEC unambigu-ously identifies it as a class I PDE (Fig 4) This PDE class includes all 11 human PDE families, and all
of its members contain the signature motif HD(LIV-MFY)xHx(AG)xxNx(LIVMFY) Their catalytic domains share 30–40% amino acid sequence identity between families [17] The overall sequence of the TcPDE-FYVE catalytic domain conforms well with that of other class I PDEs It shares between 24 and 33% of amino acid sequence identity with the 11 human PDE families, and 45 and 57% sequence iden-tity with its putative orthologs in L major (lmjPDEC) and T brucei (TbrPDEC; unpublished data) All resi-dues that have been identified as important for sub-strate recognition, selectivity and catalysis in the human PDEs [27,28] are conserved in TcrPDEC with respect to HsPDE4B2 (Fig 4) The two metal binding sites are predicted to be formed by residues H368, H372,
H409, E410, His413, N418, L438, D439, E481, M482 and
E521[28] The hydrophobic pocket that accommodates the purine moiety of the substrate is also conserved and predicted to consist of Y367, I522, A524, S525, A532,
W535, L536, I538, L539, G559, S564, V566, S569, Q570, and
F573 Interestingly, of the two residues in this pocket that contribute to selectivity for cAMP over cGMP
in HsPDE4, only Q570 (corresponding to Q443 in HsPDE4B2) is conserved, while the residue corres-ponding to N395 in HsPDE4B2 is substituted by A524 The side chain of this N395 in the structure of HsPDE4B forms two hydrogen bonds with adenine, with the 6-NH2 atom and with the 7-N ring atom, while it might only form a single interaction with either the 7-N atom or the 2-NH2 atom of guanine While the presence of an N residue at this position favors cAMP binding, it does not preclude cGMP binding However, in several of the human PDEs that accept cGMP as a substrate, the position of N395 is substituted by an alanine (HsPDE5 and HsPDE6) or glycine (HsPDE3 [27]) The presence of an alanine resi-due at this position implies that TcrPDEC might be capable of using cGMP as a substrate This could be experimentally confirmed (see below) Compared with the set of 19 residues that are completely conserved
in all 11 human PDEs [27], four substitutions in
Fig 3 Dimeric structure of the FYVE-type domain (A) Gel filtration
(Superdex 75 PC 3.2 ⁄ 30) of an N-terminal fragment of TcrPDEC
containing the FYVE-variant domain through the coiled-coil region
(amino acids 1–272) (B) Gel filtration of FYVE-type domain alone
(amino acids 1–74 of TcrPDEC) Elution positions of the
FYVE-con-taining polypeptides are indicated by asterisks Vertical black arrow:
elution position of TEF protease (27 kDa) Elution position of
molecular weight markers (open arrows): 1, aldolase (158 kDa); 2,
bovine serum albumin (67 kDa); 3, chymotrypsingen A (25.0 kDa);
4, aprotinin (6.5 kDa); 5, vitamin B12 (1.35 kDa).
Trang 5TcrPDEC are notable In the predicted helix 9, a
con-served alanine is substituted by S429 in TcrPDEC In
predicted helix 10, the first of the two vicinal histidines
is replaced by L441, and in predicted helix 11, a
con-served alanine is substituted by H479 Finally, in
pre-dicted helix 14, a conserved aspartate is replaced by
E547 The three substitutions represented by L441, H479
and E547 are specific for TcrPDEC Another class1
PDE of T cruzi, TcPDE1, that has a low Km and is
cAMP-selective [14], conforms to the mammalian PDE
pattern in all three positions These three substitutions
in TcrPDEC may contribute to its dual-substrate
spe-cificity, and⁄ or to its relatively high Km for both
sub-strates (see below)
Functional complementation of a PDE-deficient
S cerevisiae strain Deletion of the two PDE genes ScPDE1 and ScPDE2 from the S cerevisiae genome leads to an accumula-tion of cAMP in the cells, leading to marked heat-shock sensitivity [29] Heterologous complementation
of the heat-shock sensitivity phenotype of PDE-defici-ent yeast strains has proven to be a highly sensitive functional validation for suspected PDE genes [20–32] The full-size open reading frame of TcrPDEC, as well
as the predicted catalytic domain (T291–S657) were expressed in the PDE-deficient S cerevisiae strain PP5 [31] In addition, the same two constructs carrying
Fig 4 Sequence alignment of catalytic domains Grey vertical boxes indicate residues that are identical in all human PDEs as well as in TcPDE1 and TcrPDEC; open vertical boxes indicate residues that are conserved in all 11 human PDEs and in TcPDE1, but are substituted in TcrPDEC; black dots represent residues that are necessary for positioning the catalytically important histidine residue (arrow); boxes indicate the consensus helices; asterisk and bold, asparagine residue which confers cAMP preference (N395in HcPDE4B) m, metal binding pocket;
q, Q-domain [28].
Trang 6N-terminal hemagglutinin tags were also expressed.
Transformants were patched and tested for heat shock
resistance (Fig 5) All four constructs fully restored
the heat shock resistance phenotype to the indicator
strain The results of these complementation
experi-ments established that TcrPDEC codes for a functional PDE, and that this enzyme can use cAMP as a sub-strate
Characterization of catalytic activities Soluble cell lysates were prepared from yeast strains expressing either full-length TcrPDEC or its catalytic domain only The recombinant PDE activity was entirely dependent on the presence of divalent cations, with Mg2+ providing the best activity Setting the activity observed with 2 mm MgCl2 as 100%, 2 mm MnCl2 yielded 53.5 ± 15.9% activity, 0.2 mm CaCl2
15.3 ± 1.9%, and 30 lm ZnCl2 19.5 ± 3.2% The activity obtained with 2 mm MgCl2 was slightly, but consistently, further stimulated by the presence of 0.2 mm CaCl2 (116.1 ± 13.2%) In all subsequent experiments, reactions contained 10 mm MgCl2 Michaelis-Menten kinetics were determined with cAMP or cGMP as substrates (Fig 6A,B) TcrPDEC exhibits a Kmfor cAMP of 31.6 ± 9 lm (N¼ 6) This
is significantly higher than the Kmfor cAMP observed for a previously characterized PDE from T cruzi, TcPDE1 [14] Unexpectedly, TcrPDEC also hydrolyzes cGMP with a similar Vmax, and with a Km of 78.2 ± 25 lm (n¼ 3) This dual substrate specificity might reflect the presence of the A524 residue in the sequence of TcrPDEC, replacing an asparagine residue
- heat shock + heat shock
pLT-TcPDEC
pLT-TcPDEC-cat pHA-TcPDEC
pHA-TcPDEC-cat pLT1 pd6
Fig 5 Reversal of the heat-shock sensitivity phenotype of S
cere-visiae PDE deletion strain PP5 Duplicate patches of independent
transformants of S cerevisiae PP5 incubated with (left) or without
(right) an initial heat shock at 55 C for 15 min pLT-TcrPDEC,
full-length TcrPDEC; pHA-TcrPDEC, full-full-length TcrPDEC with a
N-ter-minal hemagglutinin tag; pLT-TcrPDEC-cat, catalytic domain of
TcrPDEC (W 367 -H 597 ); pHA-TcrPDEC-cat, catalytic domain of
TcrP-DEC with an N-terminal hemagglutinin tag; pLT1, empty vector
(negative control); pd6, TbPDE1 (positive control).
Dipyridamole
Etazolate
[Inhibitor] (µM) [Inhibitor] (µM)
Trequinsin
IBMX
µM cAMP
mo
µM cGMP
MP
D
C
Fig 6 Michaelis-Menten plots (A) with
cAMP as substrate and (B) with cGMP as
substrate (C) and (D) show representative
plots of IC 50 determinations for various PDE
inhibitors (substrate concentration: 1 l M
cAMP).
Trang 7that appears to confer cAMP selectivity in the
cAMP-specific PDEs This dual cAMP-specificity is a novel feature
for a kinetoplastid PDE since all PDEs characterized
in these organisms so far are entirely cAMP selective
[13,15,16,30,32, Johner et al submitted]
Inhibitor profiling
To explore the sensitivity of TcrPDEC against a
spec-trum of inhibitors, the potency of a number of
com-mercially available PDE inhibitors was determined
using 1 lm cAMP as the substrate Most of the
com-pounds tested exhibited low potency against TcrPDEC
(Table 1), though several of them are high-potency
inhibitors of various human PDEs Only three of the
compounds tested, trequinsin, etazolate, and
dipyrida-mole, exhibited IC50 values below 10 lm (Figs 6C.D)
With human PDEs, these three compounds inhibit
different PDE families with high potency (trequisin:
HsPDE3, IC50 0.0003 lm; etazolate: HsPDE4, IC50
0.55 lm; dipyridamole: HsPDE5, IC50 0.9 lm) All
three compounds have also proven to be effective
inhibitors of various PDE families from different
kine-toplastids (15,16,30,32, Johner et al submitted] The
IC50 value of the broad-spectrum PDE inhibitor
iso-butyl methyl xanthine (IBMX) against TcrPDEC is
68 lm (Fig 6D) This potency is similar to that found
for IBMX with many of the human PDEs However,
it is considerably higher than what was found for other
trypanosomatid PDEs such as TcPDE1 [14], TbPDE1 [30] or TbPDE2 [16,32]
Discussion TcrPDEC represents a novel type of class I PDE, as defined by the sequence characteristics of its catalytic domain [17] All PDEs of this class exhibit a similar overall architecture, where the N-terminal moiety con-tains various assortments of regulatory domains and the C-terminal part hosts the catalytic domain Tcr-PDEC is a notable exception to this rule, in that its catalytic domain is localized in the middle of the poly-peptide chain (amino acids T291–S657of a total of 924) The C-terminal portion contains no recognizable func-tional domains or motifs In the amino acid sequence
of the catalytic domain, all residues that have been identified as important for substrate recognition, selec-tivity and catalysis [27,28] are fully conserved In the structure of human PDE4, two residues within the hydrophobic purine-binding pocket were shown to be crucial for purine binding and for adenine-specificity (in HsPDE4B2: Q443and N395, respectively) The pres-ence of an asparagine in this position favors the bind-ing of cAMP, but does necessarily preclude the binding of cGMP However, in several of the human PDEs that accept cGMP as their substrate, the posi-tion of N395is substituted by alanine (in HsPDE5 and HsPDE6) or by glycine (in HsPDE3) [27]
Interest-Table 1 Potency of selected PDE inhibitors All IC50values were determined at 1 l M substrate.
Inhibitor
IC50(l M )
Mammalian PDE selectivity (IC 50 l M )
PDE 6 (0.4) PDE 8 (4.5) PDE 10 (1.1) PDE 11 (1–2)
PDE 4 (0.2–0.8)
*n.s., nonselective inhibitor; † isobutyl methyl xanthine; ‡ 8-methoxymethyl-IBMX; EHNA, erythro-9-(2-hydroxy-3-nonyl)adenosin.
Trang 8ingly, the position corresponding to N395 in
HsPDE4B2 is occupied by an alanine residue (A524) in
TcrPDEC Based on the information available from
the human PDEs, this finding predicts that TcrPDEC
could accept cGMP as a substrate This prediction
could be experimentally verified Recombinant
TcrP-DEC proved to be a dual-specificity PDE, with Km
values of 32 lm for cAMP and 78 lm for cGMP,
respectively These Km values are relatively high when
compared with most of the human and trypanosomal
PDEs Nevertheless, they are similar to those of the
human dual-specificity PDE HsPDE2 (30–50 lm for
cAMP and 15–30 lm for cGMP [33])
While the observation that TcrPDEC can hydrolyze
cGMP is in perfect agreement with the prediction from
its amino acid sequence, the biological significance of
this finding is not at all clear Paveto et al have
pre-sented evidence that T cruzi might contain a cGMP
signaling pathway that can be stimulated by nitric
oxide [4] In other kinetoplastids such as L major or
T brucei, no cGMP PDE activity was detectable in
whole cell lysates (unpublished data) Database
screen-ing of kinetoplastid genomes has not so far identified
any genes for putative soluble guanylyl cyclases, as
would have been predicted by the work of Paveto
et al [4] However, one cannot exclude that one or
several of the predicted adenylyl cyclases identified in
the T cruzi and other kinetoplastid genomes might
actually function as a guanylyl cyclase In mammalian
adenylyl and guanylyl cyclases, a single amino acid
substitution can determine the substrate specificity [34]
A second unusual feature of TcrPDEC is the
pres-ence of a FYVE-type domain at its N-terminus FYVE
domains have been identified in numerous proteins,
but so far never in a PDE While some FYVE
domains of some proteins were shown to interact with
PtdIns(3)P and to locate to endosomal membranes, the
functional role of the FYVE-variant and
FYVE-rela-ted domains is less clear The FYVE-type domain of
TcrPDEC is followed by two closely spaced coiled-coil
regions Such coiled-coil domains were shown to serve
to dimerize the FYVE domains in EEA1 [26] In
con-trast, gel filtration analysis of the FYVE-type domain
of TcrPDEC has now shown that it can form a stable
dimer in the absence of the coiled-coil domains This
dimerization might enhance the interaction of the
FYVE-type domain with a target membrane On the
other hand, the dimerization will also entail a
dimeri-zation of the downstream catalytic domains, and may
thus influence their catalytic properties The coiled-coil
region of TcrPDEC may mediate an additional level
of organization, the function of which remains to be
explored The overall concept of dimerization and
possibly modulation of the activity of PDE catalytic domains via their regulatory N-terminal regions has been established for human HsPDE2 and HsPDE5, where dimerization takes place via GAF domains [35]
A similar GAF-mediated dimerization and modulation also occurs in TbPDE2B of T brucei [36] and possibly also in TbPDE2C
The overall sequence of the FYVE-type domain of TcrPDEC exhibits a smaller overall electrical charge than the canonical FYVE domains The functional consequences of this are not clear, as we have not been able to demonstrate binding of a recombinant FYVE-type domain of TcrPDEC to individual phospholipids
in a spot assay While this naive approach works with certain FYVE domains [24,25], it is certainly less than definitive, since the binding requirements for individual FYVE domains may be more complex than just indi-vidual phospholipids spotted on a solid surface If the FYVE-type domain of TcrPDEC does in fact interact with membranes, it may serve to confer a precise sub-cellular localization to the enzyme This is a most interesting possibility, as the role of subcellular local-ization assumes an ever greater significance in the emerging concepts of intracellular signal transduction [37] In fact, being in the right place at the right time may be the preeminent requirement for the proper working of a signaling enzyme such as a PDE
Experimental procedures Materials
Radiochemicals were purchased from Moravek Biochemi-cals Inc (Hartmann Analytik, Zurich, Switzerland) cAMP and cGMP (sodium salts) were obtained from Sigma (Buchs, Switzerland) PDE inhibitors were from the follow-ing sources: I I IBMX, papaverine, milrinone, trequin-sin, 8-methoxymethyl-IBMX, dipyridamole and rolipram were from Sigma; erythro-9-(2-hydroxy-3-nonyl)adenosin (EHNA), zardaverine, cilostamide and Ro-20–1724 were from BioMol (Anawa Trading, Wangen, Switzerland); etaz-olate and pentoxifylline were from Calbiochem (Juro Sup-plies, Lucerne, Switzerland) DNA sequencing reactions were run with BigDye terminators (PE-Biosystems) and were analyzed on an ABI Prism 377 instrument Genomic DNA was extracted from the T cruzi promastigotes sup-plied by R Brun, Swiss Tropical Institute, University of Basel
Identification and cloning of TcPDE-C
A search of the T cruzi database (http://www.genedb.org/ genedb/tcruzi) for PDE-coding sequences identified an open
Trang 9reading frame (Tc00.1047053506697.20) that appeared to
code for a highly unusual PDE, termed TcrPDEC Its
entire coding region was amplified by high-fidelity PCR
(Expand High Fidelity PCR System, Roche Diagnostics,
Rotkreuz, Switzerland) using T cruzi genomic DNA as the
template The primers used were TcPDE4 for (5¢-CAG
TCGACATATGTCGGAGGACGCTGGGCTTC-3¢) and
TcPDE4-rev (5¢-CGTGGATCCTCAGCACTGCGTCAA
CAGAGTG-3¢) The PCR product was cloned into the
pCR2.1-TOPO vector The predicted catalytic region of
TcrPDEC (T291–S657) was amplified separately, using
prim-ers TcPDE4-catf (5¢-CAGTCGACATATGACAATACT
CGCAGTTGTTCC-3¢) and TcPDE4-catr (5¢-CTGGAT
CCTCAACTGGCTGTTCTCAGCTCCTG-3¢) and cloned
into pGEM-T Easy plasmid vector (Promega, Catalys,
Wallisellen, Switzerland) All cloned PCR products were
verified by DNA sequencing
Expression in S cerevisiae
For expression in S cerevisiae, the entire open reading
frame and the catalytic region (T291–S657) of TcrPDEC were
cloned via SalI and BamHI restriction sites into two variants
of the yeast expression vector pLT1 [30] One variant directs
the expression of the genuine protein, while the other adds
an N-terminal hemagglutinin-tag to facilitate detection of
the recombinant proteins Transformation of the constructs
into the PDE-deficient S cerevisiae strain PP5 (MATa leu2–
3 leu2–112 ura3–52 his3–532 his4 cam pde1::ura3 pde2::
HIS3) was carried out as described earlier [30,31]
Complementation assay
The heat-shock assay to detect complementation of the
PDE-deficient phenotype of the S cerevisiae strain PP5 was
carried out exactly as described [30] Single yeast colonies
were replica patched onto YPD plates (10 gÆL Bacto-Yeast
extract, 20 gÆL Bacto-Peptone, 20 gÆL glucose, 20 gÆL
bacto-agar) prewarmed to 55C, and were incubated for another
15 min at 55C Plates were then cooled to room
tempera-ture and incubation was continued at 30C for 18–36 h
Yeast cell lysis
Yeast cell lysis was performed as described [32] with minor
modifications Briefly, yeast cells grown to mid-log to
end-log phase in SC-leu medium were collected, resuspended in
the original volume of prewarmed YPD medium, and
incu-bated for an additional 3.5 h at 30C to maximize protein
expression Cells were washed twice in H2O, and the washed
cell pellet was stored at)70 C The pellet was thawed on
ice and suspended in an equal volume of ice-cold extraction
buffer [50 mm Hepes pH 7.5, 100 mm NaCl, 1· Complete
protease inhibitor cocktail with EDTA (Roche
Diagnos-tics)] Cells were lysed by grinding with glass beads (0.45– 0.50 mm) in 2 mL Sarstedt tubes, using a FastPrep FP120 cell disruptor (3· 45 s at setting 4) The subsequent centrif-ugation steps were carried out exactly as described Glycerol was added to the resulting supernatant to a final concentra-tion of 15% (v⁄ v), aliquots were snap-frozen in liquid nitro-gen and were stored at )70 C Protein expression and stability of the enzyme under assay conditions were monit-ored by immunoblotting, using a monoclonal antibody against the hemagglutinin tag (Roche Diagnostics)
Protein–lipid overlay assay Protein–lipid binding studies were performed as described [38], with the following modifications: Lipids were dissolved
in chloroform⁄ methanol ⁄ water (1 : 2 : 0.8 by volume) A 20–2500-pmol amount of each lipid were spotted onto nitrocellulose filters and were air-dried for 1 h Membranes were blocked with 3% (w⁄ v) fat-free bovine serum albumin (Sigma) in 10 mm Tris⁄ HCl, pH 8.0, 150 mm NaCl, 0.1% (v⁄ v) Tween-20) for 1.5 h at room temperature Filters were incubated with 2 lgÆmL)1 of recombinant GST-fusion pro-teins overnight at 4C After washing the filters five times with incubation buffer, the bound GST fusion protein was quantitated by developing the filters with GST body, followed by horse radish conjugated IgG anti-body
Phosphodiesterase assay PDE activity was determined by a modification of the two-step procedure of Thompson and Appleman [39] as des-cribed previously (Johner et al submitted) PDE activity was determined in 50 mm Hepes pH 7.5, 0.5 mm EDTA,
10 mm MgCl2, 50 lgÆmL)1bovine serum albumin in a final volume of 100 lL Each assay contained 50 000 cpm
3
H-labelled cAMP or cGMP, with unlabeled cAMP or cGMP added to the desired total substrate concentration Reactions were run at 30C for 15 min They were then stopped by the addition of 25 lL of 0.5 m HCl, neutralized with 20 lL 1 m Tris base, and digested with 10 lL of calf intestinal alkaline phosphatase (Roche Diagnostics; 1 unit per 10 lL) for 15 min at 37C After dephosphorylation, the reactions were applied to 1 mL columns of QAE-Sepha-dex A25 in 30 mm ammonium formate, pH 6.0 The
3H-adenosine or 3H-guanosine formed during the reaction was eluted with 1.6 mL of 30 mm ammonium formate,
pH 6.0 into 3.5 mL water-miscible scintillation fluid (Pack-ard Ultima Flo; Pack(Pack-ard Bioscience, Zurich, Switzerland)
In all reactions no more than 20% of the substrate was hydrolyzed Assays were always carried out in triplicates, and at least three independent experiments were performed
IC50studies were all carried out at 1 lm substrate concen-tration Inhibitors were dissolved in dimethyl sulfoxide
Trang 10The dimethyl sulfoxide (DMSO) concentration in the
final assay solutions never exceeded 1%, and appropriate
control reactions with DMSO alone were always included
Data were analyzed using the GraphPad Prism software
package
Gel filtration analysis of the FYVE-variant domain
PCR-amplified DNA fragments corresponding to amino
acids 1–74 of TcrPDEC (FYVE-variant domain alone) and
1–272 (FYVE-variant domain plus coiled-coil region) were
cloned into the expression vector pGST-parallel2 [40]
The recombinant proteins are N-terminally fused to
glutathione-S-transferase, from which they are separated by
a TEV protease cleavage site Recombinant protein from
125 mL bacterial culture were purified by batch-adsorption
to 125 lL Glutathione-Uniflow resin (BD Biosciences,
Basel, Switzerland) for 2 h at 4C The resin was washed
four times with ice-cold 50 mm Tris⁄ HCl, pH 7.5, 150 mm
NaCl, 10 mm 2-mercaptoethanol, 5 mm sodium citrate,
10 lm ZnCl2 and 5% glycerol After washing, the beads
were suspended in an equal volume of washing buffer,
30–50 units of AcTEV protease (Invitrogen; Juro Supplies,
Lucerne, Switzerland) were added, and the slurry was
incu-bated for 36 h at 6C on a rotating wheel The beads were
then pelleted by centrifugation, and the supernatant
con-taining the released FYVE-variant domain was purified by
centrifugation through a 0.2 lm filter
Proteins were analyzed on a Superdex 75 PC 3.2⁄ 30 gel
filtration column, using the Pharmacia Smart system The
column was precalibrated with the following markers:
aldo-lase (158 kDa), bovine serum albumin (67 kDa), ovalbumin
(44 kDa), chymotrypsinogen A (25 kDa), RNase A
(13.7 kDa), aprotinin (6.5 kDa) and vitamin B12 (1.35 kDa)
All fractions were subsequently analyzed by gel
electrophor-esis The larger FYVE-variant⁄ coiled-coil polypeptide was
further analyzed on a Superdex 200 PC 3.2⁄ 30 column
calib-rated with the same markers, plus the following additional
ones: thyroglobulin (670 kDa), ferritin (450 kDa) and
cata-lyze (232 kDa)
Acknowledgements
We would like to thank M Linder and X Lan Vu for
their technical assistance, R Brun of the Swiss
Trop-ical Institute, University of Basel, for T cruzi cultures,
the laboratory of J H Hurley (NIH, Bethesda, MD,
USA) for providing expression plasmids for
Vps27p-FYVE-GST fusion proteins, Peter Buetikofer (Institute
of Molecular Biology, University of Bern) for
provi-ding phospholipids and valuable advice This work
was supported by grant Nr 3100–067225 of the Swiss
National Science Foundation, and by the COST B22
programme of the European Union
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