Bariloche protein symposium argentine society for biochemistry and molecular biology

152
BIOCELL, 27 (Suppl. I), 2003
LI-P18.
LIPID DYNAMICS IN DISCOIDAL LIPOPROTEIN
COMPLEXES OF APOLIPOPROTEIN A-I
Prieto ED
1
, Oliviera T
2
, Lamy MT
2
, and Garda HA.
1
1
INIBIOLP (CONICET-UNLP). Facultad de Ciencias Médicas,
Calles 60 y 120, 1900-La Plata; and 
2
Instituto de Física,
Universidade de São Paulo, São Paulo, SP, Brazil. E-mail:
[e-mail]
Fiscoidal high density lipoprotein complexes (dHDL) of
apolipoprotein AI (apoAI) are intermediates in HDL metabolism
and reverse cholesterol transport. Although dHDL formation “in
vivo” requires the assistance of cellular proteins, they can be
obtained “in vitro” by mixing apoAI with vesicles of zwitterionic
phospholipids at the gel to liquid-crystalline phase transition
temperature (Tt). several models for dHDL were proposed which
differ in the arrangement of apoAI helices, but all of them agree
that the protein is wrapped around the edge of a phospholipid
bilayer. in this work, we used electronic paramagnetic resonance
(EPR) to obtain information about how the dynamics of disc lipids
is affected by apoAI. different spin-labeled phospholipids probes
reporting the mobility at different bilayer regions were used:
TEMPO-PC (with label in the polar head) as well as 5- and 16-
doxyl PCs (with label in the hydrocarbon chain). results show
that above Tt, apoAI discs have a decreased hydrocarbon chain
mobility (reported by 5- and 16-doxyl PCs) and an increased
mobility of polar head groups (sensed by TEMPO-PC) compared
with protein-free bilayers. the opposite effect of apoAI is observed
below Tt. these results are typical for proteins inserted or spanning
the bilayer and are discussed taking into account the proposed
models for dHDL
LI-P19.
EFFECT OF APOLIPOPROTEIN A-I ON OLEIC ACID
ESTERIFICATION IN CELL CULTURED FIBROBLASTS
Gonzalez MC, Igal RA, Tricerri MA, and Garda HA.
INIBIOLP (CONICET-UNLP). Fac. de Ciencias Médicas. Calles
60 y 120. 1900-La Plata. E-mail: [e-mail]
Apolipoprotein A-I (apoAI) is the main protein of high density
lipoproteins (HDL) and plays a key role in several steps of reverse
cholesterol transport, as lipid removal from peripheral cells,
activation of LCAT, and interaction with cellular proteins (ABCAI,
SR-BI). It was proposed that apoAI and HDL trigger different
signals in peripheral cells. Some of them result in mobilization
toward the plasma membrane of intracellular cholesterol depots,
as the one available to esterification by acylCoA-cholesterol acyl
transferase. Metabolic events taking place in this process are still
matter of debate, involving cell type and metabolic rate, as well
as characteristics of cholesterol acceptors. On the other hand,
nothing is known about apoAI domains involved in these cellular
responses. The ability to decrease cholesterol esterification and
exerting cholesterol efflux were measured for apoAI and HDLs in
two cell types: a normal cell line of human skin fibroblasts
(CCD27) and other transformed with SV40 (WI38). Results show
that lipid-free apoAI stimulus decreases cholesterol esterification
in CCD27 but not in WI38 cells. The possible implication of a
central apoAI domain was investigated by using a synthetic peptide
and two apoAI mutants in which the central region is replaced or
changed in orientation. Lack of response to apoAI stimulus in
transformed cells is discussed as well as the involved apoA-I
domains.
LI-P20.
ON THE CENTRAL APOLIPOPROTEIN A-I MEMBRANE-
INSERTING DOMAIN
Toledo JD, Tricerri MA, and Garda HA.
INIBIOLP (CONICET-UNLP). Facultad de Cs. Médicas. Calles
60 y 120. 1900-La Plata. E-mail: [e-mail]
We have previously reported that apolipoprotein A-I (apoAI) in
reconstituted high density lipoproteins (rHDL) interacts with
membranes through the insertion of a small central domain located
between residues 87 and 112 (J Biol Chem 2001). Recent studies
using a synthetic peptide (communicated in SAB 2002), indicated:
a) that this domain is responsible for the selectivity of apoAI for
cholesterol containing membranes, and for promoting membrane
cholesterol desorption; and b) that the membrane-inserted state is
oligomeric. This last fact and recent evidences indicating that
central regions of both apoAI molecules are close together in a
rHDL disc, allowed us to hypothesize that an intermolecular helix
bundle forms the active membrane inserting domain. We report
here additional information on the membrane-inserted state of this
region obtained by fluorescence resonance energy transfer (FRET)
and fluorescence quenching measurements. Results indicate that
at least one of the four apoAI Trp residues (presumably Trp108)
locates at a distance short enough from the vesicle bilayer to give
measurable FRET toward a phospholipid acceptor (DPH-PC)
located in the vesicles. However, no Trp residue would be present
in the membrane-inserted region since no detectable Trp
fluorescence quenching was produced by vesicles containing spin-
labeled phospholipids with the quencher group either at the polar
head (TEMPO-PC) or in the hydrophobic region (5-, 7-, or 12-
doxyl PC).
LI-P21.
LIPID METABOLISM IN HEP G2 CULTURED CELLS
TREATED WITH  HMG-CoA REDUCTASE INHIBITORS
Polo, Mónica; Bellini, María José; Bravo, Margarita G. de
INIBIOLP (UNLP-CONICET) Facultad de Cs. Médicas. La Plata
E-mail: [e-mail]
HMG-CoA reductase inhibitors like statins and monoterpenes
diminish cellular cholesterol content. This fact increases the
proteolysis of the sterol regulatory element binding protein
(SREBP), which bind to sterol regulatory elements contained in
promoters of genes involved in the synthesis of LDL receptors
and key enzymes of cholesterol and fatty acid metabolism. We
investigated the alterations produced by simvastatin (a statin) and
geraniol (a natural monoterpene) on lipid metabolism in Hep G2
cells. Cells were treated with 1
µM simvastatin (S), 10 µM geraniol
(G) or 1
µM simvastatin + 10 µM geraniol (SG)  for 24 h. Three
hours prior to starvation, 
14
C-acetate (1
µCi/ml) was added. Total
lipids were extracted and 
14
C incorporation in non saponifiable
lipids, fatty acids, neutral  and phospholipids were determined.
All treatments incremented 
14
C incorporation in total lipids and
in fatty acids. S and SG groups showed a greater 
14
C incorporation
in all phopholipid fractions, G group did not show and increment
in the incorporation in PC. All groups showed a diminished 
14
C
incorporation in cholesterol, S group also in lanosterol and
squalene, but G group increased its incorporation in squalene.
These results demonstrated that all treatments augmented fatty
acid synthesis and diminished cholesterol synthesis. Geraniol is
likely to inhibit PC synthesis due to the accumulation of an
intermediate metabolite as a consequence of an inhibition of the
conversion of squelene into cholesterol.

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