Biology Reference
In-Depth Information
cat
A
GAF-A
GAF-B
cat
B1, B2
*
FYVE
CC
cat
C
cat
D
Fig. 2 Schematic representation of the four kinetoplast PDE families.
Cat
class 1 catalytic
domain,
GAF-A, GAF-B
GAF domains of PDEB1 and PDEB2,
FYVE
N-terminal FYVE domain
of PDEC,
CC
coiled-coiled domain,
asterisk
predicted conserved PKA phosphorylation site
between the GAF domains,
black dots
phosphorylated serine residues at the C-terminus of
TbrPDEB1
L. tarentolae
;
http://tritrypdb.org/tritrypdb/
) contain the same set of four class I
PDEs (Fig.
2
and Table
1
). PDEA, PDEC, and PDED are all single-copy genes in
T. brucei
and
Leishmania
(see Table
1
), with
T. cruzi
representing a special case.
The
T. cruzi
CL Brener strain that was used for genome sequencing is a hybrid, so
that many single-copy genes are actually present in two versions that reflect their
different ancestry (Weatherly et al.
2009
). For PDEB, all the above genomes
contain two tandemly linked, very similar, but nonidentical copies. The PDEs
coded by these two genes, PDEB1 and PDEB2, exhibit clearly distinct subcellular
locations (see below). A unified nomenclature for the kinetoplastid PDEs has been
proposed (Kunz et al.
2006
); an updated version is given in Table
1
.
PDEA
. TbrPDEA was the first trypanosomatid PDE to be cloned, expressed, and
characterized (Kunz et al.
2004
). Subsequently, orthologues of TbrPDEA were
identified in all kinetoplastid genomes. They are all single-copy genes, with the
exception of
T. cruzi
where two copies are present and share between 40 and 60%
amino acid sequence identity. The
T. brucei
gene (formerly designated TbPDE1)
was functionally identified as a cAMP-PDE by complementation screening of a
T. brucei
cDNA library in the yeast
Streptomyces cerevisiae
(Kunz et al.
2004
): it is
a single-copy gene located on chromosome 10, which codes for a protein of 620
amino acids. Constitutively low levels of mRNA are present both in bloodstream
and in procyclic forms. The class 1 catalytic domain of TbrPDEA is situated in the
C-terminal region (M
285
-Q
618
) and exhibits cAMP-selectivity, with an unusually
high
K
m
for cAMP of
600
m
M. The enzyme is fully resistant to most commercial
PDE inhibitors, but is inhibited by sildenafil and trequinsin with approximate
K
i
values of 1.0-2.5
m
M, respectively. Very similar results were also obtained with its
T. cruzi
homologue, TcrPDEA1, which exhibits a
K
m
for cAMP of 190
m
M, is not
affected by cGMP, and complements a PDE-deficient yeast strain (Alonso et al.
2007
).
For
L. major
, the gene for LmjPDEA was also shown to complement a PDE-
deficient strain of
S. cerevisiae
(Johner et al.
2006
). A common observation for the
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