Bariloche protein symposium argentine society for biochemistry and molecular biology

38
BIOCELL, 27 (Suppl. I), 2003
LI-C3.
PHOSPHATIDYLCHOLINE SYNTHESIS REGULATION
BY PGD
2
 IS MEDIATED BY MAPK ACTIVATION
Fernández-Tome M.; Favale N.; Speziale E.; Sterin-Speziale N.
Cátedra de Biología Celular. Facultad de Farmacia y Bioquímica.
UBA. IQUIFIB-CONICET. E-mail: [e-mail]
Phosphatidylcholine (PC) is a main lipid of biomembranes with
important structural and functional roles. In previous works, we
have demonstrated that renal papillary PC synthesis is regulated
by endogenous-synthesised prostaglandin D
2
 (PGD
2
) and that such
regulation occurs on PC biosynthetic enzymes. The purpose of the
present work was to evaluate the mechanism by which PGD
2
 exerts
its modulatory action on PC synthesis. We first studied which
receptor could be activated by PGD
2
 interaction and found that
sulprostone (a PGE
2
 receptor, EP3 full-agonist) mimicked the effect
of PGD
2
 on PC synthesis. Hence, we studied if MAPK activation,
which was reported to be involved in EP3 actions, was mediating
PGD
2
 regulatory action on papillary PC synthesis. Thus, we
evaluated renal papillary PC biosynthesis as 
32
Pi incorporation to
PC in the absence or in the presence of PGD
2
 with or without the
addition of U0126, a selective MEK inhibitor. We found that PGD
2
induced MAPK-phosphorylation which was prevented by U0126.
This inhibitor also blocked the PGD
2
 effect on PC biosynthesis
with the same pattern observed for MAPK activation, thus
suggesting the involvement of this pathway in the maintenance of
PC synthesis in renal papillary membranes.
LI-C4.
HEAT INDUCED CHANGES IN TESTICULAR LIPIDS
CONTAINING VERY LONG CHAIN POLYUNSATURATED
FATTY ACIDS (VLCPUFA)
Furland, N.E.; Maldonado, E.N. and Aveldaño, M.I.
INIBIBB, CONICET-UNS, 8000 Bahía Blanca, Argentina. E-mail:
[e-mail]
Hyperthermia is known to result in adverse consequences for the
normal adult testis of several mammals, including a reduction of
testicular weight and a period of partial or complete infertility,
since it selectively damages germ cells. We observed previously
that experimental cryptorchidism in rats resulted in a marked
reduction of germinal cells, concomitantly with a decreased amount
of testicular lipids containing polyunsaturated fatty acids (PUFA)
with long and very long chains. In this work we aimed at studying
the long-term effects on lipids of a single exposure (15 min) of
the testis to moderate heat (43ºC). Four days after the heat shock,
there was a significant increase in the amount of cholesterol esters
and 1-alkyl-2,3 diacylglycerols. The fatty acid profile of both
showed a slight increase in pentaenoic PUFA (22:5n-6) and a
concomitant decrease of pentanoic VLCPUFA (especially 28:5n-
6). The earliest effect observed (6 hours posttreatment) was a
significant, apparently transient, increase in the amount of
ceramides (Cer). The increased Cer showed a peculiar fatty acid
pattern, with an increase in fatty acids other than VLCPUFA. After
four days, the proportions of PUFA and VLCPUFA were
significantly reduced, and after 10 days the VLCPUFA were
virtually absent from the Cer remaining in the testis.
LI-C5.
GLUCOSE-INDUCED DECREASE OF PHOSPHATIDYL-
ETHANOLAMINE AFFINITY FOR THE TRANS-
MEMBRANE SURFACE OF MEMBRANE PROTEINS
Levi, Valeria; Villamil Giraldo, Ana M.; Castello, Pablo R.; Rossi,
Juan P.F.C. and González Flecha, F. Luis.
IQUIFIB, FFYB, Universidad de Buenos Aires, Argentina. E-mail:
[e-mail]
Integral membrane proteins span biological membranes defining
a lipid-protein interface. The amphiphiles located in contact with
the protein surface have a restricted mobility as a consequence of
interactions with surface residues of the transmembrane domain.
Hence, the transmembrane segments detect modifications in the
bilayer and induce changes on protein conformation, which -in
some cases- are translated into functional changes. We have
recently developed a simple resonance energy transfer method to
quantify amphiphile-membrane protein interactions [Levi et.al.,
Anal Biochem 317: 171-179, 2003], which uses a phospholipidic
fluorescent probe that competes with the unlabeled amphiphiles
for positions on the transmembrane surface of the protein. In this
study, the plasma membrane calcium pump was reconstituted in
mixed micelles composed of the detergent C
12
E
10
,
phosphatidylcholine and phosphatidylethanolamine (PE) or PE
preincubated with glucose. The protein fluorescence spectrum was
registered at different mole fractions of the micelle components,
and the exchange constants were determined being 1.8 ± 0.2 for
the control and 1.0 ± 0.1 for glucose treated lipids. These values
indicate that incubation of PE with glucose produce a significant
decrease of PE-affinity for the transmembrane domain of the
protein. Similar results were obtained using the Na,K-ATPase
reconstituted in the same micellar systems.
LI-C6.
c-FOS REORGANIZES PHOSPHOLIPIDS AT THE
INTERFACE
Graciela Borioli, Carla Rosetti and Bruno Maggio.
Facultad de Ciencias Químicas, Universidad Nacional de
Córdoba. E-mail: [e-mail]
The transcription factor c-Fos has recently been studied from
viewpoints unrelated to nuclear transcription. It has been shown
that it activates phospholipid biosynthesis in vivo and in vitro
through a citoplasmic activity associated with endoplasmic
reticulum. It also is tensioactive,  interacts with phospholipids,
especially phosphatidyl inositoldiphosphate (PIP
2
), and undergoes
two reversible molecular reorganizations in response to lateral
pressure changes in monolayers. Furthermore, it modulates PLA
2
,
sphingomyelinase and PLC activity on lipid monolayers in a finely
tuned way depending on surface lateral pressure. Aimed at
elucidating the mechanism by which c-Fos could play a role in
phospholipid metabolism, we analyse the compressional behavior
of lipid-protein film mixtures seen by surface lateral pressure and
surface potential monitoring, epifluorescence and Brewster angle
microscopy. We find that c-Fos affects phospholipid intermolecular
organization. Mixed films with either PIP
2
 or
dilauroylphosphatidylcholine (PC) are respectively condensed or
expanded. The contribution to deviation from ideality of each
component in the mixture depends on the film’s packing and
composition. In addition, mixtures with PIP
2
 show domain
segregation and shifts in both of the protein’s interfacial
reorganizations, while those with PC are largely homogeneous with
protein reorganizations that remain independent of the lipid. The
capacity of c-Fos to affect interface structuring according to packing
state, lipid nature and the protein’s own proportion, further support
the idea that it could participate as a signal transducer in membrane
function.

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