Bariloche protein symposium argentine society for biochemistry and molecular biology

151
BIOCELL, 27 (Suppl. I), 2003
LI-P14.
INTERACTIONS BETWEEN C-FOS AND C-JUN
Del Boca Maximiliano, Borioli Graciela A.
CIQUIBIC, Departamento de Química Biológica, Facultad de
Ciencias Químicas, U.N.C. Cordoba, Argentina. E-mail:
[e-mail]
The early expression transcription factors c-Fos and c-Jun can form
heterodimmers, but only c-Jun homodimmers complexes called
activator protein 1 (AP-1) by a leucin zipper domain. AP-1 binds
to DNA by a basic domain and regulates processes like cell
proliferation and differentiation. These results have been obtained
either working with cellular extracts and co-immunoprecipitation,
or measuring interaction in vitro between short c-Fos and c-Jun
peptides that include the leucine zipper and the binding domains,
by crystallographic analysis, resonance energy transfer, shifts and
footprint analysis, and column molecular filtration.  Recent studies
show that c-Fos and c-Jun can form stable Gibbs and Langmuir
monolayers in an air-buffer interface, and they generate an
increment in the surface lateral pressure similar to the increments
generated by membrane proteins. c-Fos and c-Jun can also
penetrate differently in phospholipid monolayers depending on
polar head group of the phospholipid. In vivo studies show that c-
Fos regulates phospholipid biosynthesis in association with the
endoplasmic reticulum membrane. In a simple system with
recombinant complete or deletion mutant proteins, we have used
electrophoresis in native gels, purification in molecular exclusion
columns, cross-linking analysis, immunoprecipitation, and
monolayer techniques to explore c-Fos and c-Jun interactions. We
show that c-Fos and c-Jun don’t form stable hetero or homodimmers
complex in solution, although they have the ability to interact in
monolayers.
LI-P15.
IDENTIFICATION OF A LEISHMANIA MAJOR Ä9 FATTY
ACID DESATURASE BY HETEROLOGOUS EXPRESSION
IN SACCHAROMYCES CEREVISIAE
Guillermo A. Petrini, Silvia G. Altabe and Antonio D. Uttaro.
IBR-CONICET-Dto. de Microbiología, Fac.Cs. Bioqs. y Farms.,
UNR, Suipacha 531, 2000-Rosario, Santa Fe, Argentina. E-mail:
[e-mail]
Trypanosomatids  have a high proportion of long-chain
polyunsaturated fatty acids (22:5, 22:6), linoleic acid (18:2) and
α-linolenic acid (18:3). The presence of these molecules suggests
that fatty acid desaturation occurs via the “plant pathway”. It was
confirmed with our previous work, where we isolated and
characterized an oleate desaturase from T. brucei, which is a key
enzyme in polyunsaturated fatty acid biosynthesis. These
differences on the degree and type of desaturation between the
trypanosomatids and their mammalian hosts, make desaturases a
good target for chemotherapeutic drugs. L. major 
∆9 desaturase
gene (des9) was identified, cloned and sequenced. It is the first
enzyme in the biosynthesis of unsaturated fatty acids. Des 9 gene
encoded a protein with a high degree of similarity to 
∆9 desaturases
from fungi and contained the three conserved histidine boxes, a
C-terminal cytochrome b
5
 domain and transmembrane domains
characteristic of endoplasmic reticulum membrane-bound 
∆9-
desaturases. The enzyme was functionally characterized by
complementation of an ole1  Saccharomyces cerevisiae mutant.
Fatty acid analysis of yeast transformants expressing the pLmDes9
showed an elevated level of oleic acid (18:1) compared to
palmitoleic acid (16:1). A detailed biochemical characterization
is shown.
LI-P16.
ROLE OF  L-FABP ON THE INCORPORATION OF
SATURATED AND POLYUNSATURATED FATTY ACIDS IN
ENDONUCLEAR LIPID POOLS
Sabina M. Maté
1
, Rodolfo R. Brenner
1
 and Ana Ves-Losada
1,2
.
1
INIBIOLP, Facultad de Ciencias Médicas, UNLP, calles 60 y 120,
1900, La Plata. 
2
Facultad de Ciencias Exactas, UNLP. E-mail:
[e-mail]
Our aim was to study the role of L-FABP on the trafficking of
fatty acids from cytosol to nuclear lipid pools where they regulate
the expression of certain genes. Nuclei and nuclear matrix (Mx:
nuclei without membrane) of rat liver cells were incubated in vitro
with [1-
14
C]18:0 and [1-
14
C]20:n-6, either free or bound to the L-
FABP. Incorporation and esterification of 18:0 and 20:4n-6 were
carried out in nuclear and endonuclear lipid pools. We observed
that the incorporated total radioactivity was not modified by the
L-FABP. Firstly, [1-
14
C]18:0 and [1-
14
C]20:4n-6 were incorporated
into FFA>PL>>TAG (in the presence of ATP and CoA). Then, [1-
14
C]fatty acid incorporated was decreased in FFA, increased in
TAG, and saturated in PL, with the incubation time. In endonuclear
pools (Mx) 18:0 and 20:4n-6 bound to L-FABP were incorporated,
showing a greater proportion in DAG and PI, respectively. In
conclusion, 18:0 and 20:4n-6, exogenous, free or bound to L-
FABP,are incorporated into total nuclear and endonuclear lipid
pools through an acyl-CoA pathway. L-FABP does not affect nuclear
FA uptake, but estimulates the FA esterification, directing the fatty
acids into specific lipid fractions.
LI-P17.
HOW  DOES APOLIPOPROTEIN A-I  INTERACT WITH
MEMBRANES?
Arnulphi, Cristina; Tricerri, M. Alejandra; Sanchez, S.; Gratton,
Enrico.
FaMAF, Universidad Nacional de Córdoba; INIBIOLP, La Plata,
Argentina and Laboratory for Fluorescence Dynamics, Illinois,
USA. E-mail: [e-mail]
In a previous work we investigated by using calorimetric and
optical techniques, the interaction of apolipoprotein A-I with model
membranes of various lipid composition. The dipolar relaxation
of the fluorescent probe LAURDAN allows the calculation of the
General Polarization (GP). This function lets infer the lipid phase
of the membranes. We observed that inclusion of Sphingomyelin
(SM) molecules in bilayers of pure phosphatidilcholine (POPC)
at T< 32°C induced dynamical restriction of lipid environment
(increasing of GP), but yielded the contrary effect at higher
temperatures. This phenomenon correlates with the formation of
lipid domains observed by Two Photon Fluorescence Microscopy
in the same range of temperatures. The study of lipid membranes
containing cholesterol (POPC/SM/CHOL) (similar to lipid rafts)
gave higher values of GP, showing a change of the lipid phase
towards to a gel state. The addition of the protein to the investigated
membranes showed a differential behavior depending of the lipid
composition of the membrane and the temperature at which the
reaction was performed. In membranes of pure POPC at T> 22°C,
and of POPC/COL at T> 37°C, the binding of the protein induces
a higher fluidity of the membrane. By the contrary, in SM-
containing membranes we measured higher values of GP for both,
POPC/SM bilayers (at T> 30°C) and for POPC/COL/SM (at T>
15°C). These results show that the composition and the physical
state of the membrane play an important role during the interaction
of apoA-I with membranes.

---