trans

252 INTRACELLULAR SIGNALING
domain), or ‘atypical’ (are insensitive to Ca 2
or diacylglycerol). Ca 2 binding is mediated by
a cleft structure referred to as the C2 domain.
Other proteins, such as PLC- and synaptotagmin,
also utilize a similar structure to bind
Ca 2 . In mammalian cells, PKC isoforms have
a wide range of targets, and these are generally
responsible for the sustained phase of the
Ca 2 response. Artificial and endogenous substrates
have been used to detect PKC-like
activity in cell fractions from T. brucei, T. cruzi,
Giardia and Entamoeba. Surprisingly, no member
of the PKC family has been cloned from
parasitic protozoa. The activity of PKC is
determined in part by its distribution in the
cell. Mammalian cells utilize a signal anchor
protein (the Receptor for Activated C-Kinase,
or RACK1) as a scaffold for signal complex
formation. RACK1 is a beta-propeller protein
with a structure that is similar to G of heterotrimeric
G proteins. Homologs of RACK1 have
been identified in T. brucei, Leishmania and
Crithidia. The binding partners for parasite
RACK1 are not known, but in T. brucei, mRNA
levels for RACK1 are elevated in G o arrested
stumpy cells and in cells induced to undergo
an apoptosis-like cell death.
EF-hand Ca 2 -binding proteins
Within parasitic protozoa a great diversity in
EF-hand Ca 2 -binding proteins has been
described (Figure 11.3A, B). The EF-hand was
originally identified by X-ray crystallography
of muscle parvalbumin. The motif involves two
alpha helices, separated by a Ca 2 -binding loop
(helix-loop-helix motif). The X, Y, Z,X, Y, Z
coordination sites are residues 1, 3, 5, 7, 9 and
12 within the loop. Water within the loop contributes
a seventh coordination site, although
in parvalbumin this is not the case and instead,
the amino acid in the Z position (Glu) uses
both of its oxygens to bind Ca 2 . The result is a
pentagonal bipyramidal organization of Ca 2
coordination within the loop.
Calmodulin is the most widely distributed
of the EF-hand motif proteins (Figure 11.3A).
Calmodulin contains four EF-hand motifs that
change conformation upon binding Ca 2 and
expose a central helical region that serves as an
interaction site for target proteins. Calmodulin
is present in all eukaryotic cells and has been
cloned from T. brucei, T. cruzi, and P. falciparum.
Inhibitors of calmodulin, including
the naphtholenesulfonamides (W7) along with
the phenothiazines (trifluoperazine and chlorpromazine)
have deleterious effects on T. brucei,
T. cruzi, T. gondii, Plasmodium, Giardia and
Entamoeba. In T. brucei, the calmodulin gene
is contained within a locus of four tandem
repeats. Knockout of one locus slows trypanosome
growth, while knockout of both loci
cannot be achieved, suggesting that calmodulin
is essential to these cells. Calmodulin has
no enzymatic activity of its own, and instead
modulates the activity of target proteins in
response to Ca 2 . As a consequence, the identification
of calmodulin-binding proteins is an
important step towards understanding Ca 2
regulation in parasitic protozoa. Slight differences
in gene sequence between the various
parasite calmodulins may allow interactions
with unique parasite-specific target proteins.
However, this hypothesis has yet to be verified.
In T. brucei, elongation factor-1 (EF-1) has
been identified as a calmodulin-binding protein.
The interaction between calmodulin and
EF-1 has been confirmed in plants and mammals.
Calmodulin-binding may coordinate the
many activities of EF-1 including its ability to
regulate protein translation, cytoskeletal organization,
inositide metabolism and proteasome
activity. Calmodulin also appears to associate
with the paraflagellar rod. Whether calmodulin
contributes to bending of the rod is not known.
However, in T. cruzi, a calmodulin-sensitive
BIOCHEMISTRY AND CELL BIOLOGY: PROTOZOA