Download Structural and multifunctional properties of cardiac fatty acid

Biochemical Society Transactions
Structural and multifunctional properties of cardiac fatty acid-binding protein:
from fatty acid binding to cell growth inhibition
F. Spener and T. Borchers
Department of Biochemistry, University of Munster, Munster, Germany
806
Introduction
The cardiac fatty acid-binding protein is one type of
a family of nonenzymic proteins [ I , 21 that bind
hydrophobic ligands. The localization of these fatty
acid-binding proteins (FAHPs) is mainly cytosolic
kvith concentrations up to - 5% 13I of soluble proteins. A closer inspection of the subcellular distribution by means of immunohistochemical methods
and e.1.i.s.a. [ 41, however. reveals a type-specific
compartmentation of FAHPs, e.g. the cardiac-type
FAHP is associated with myofibrils and mitochondrial matrix, whereas in hepatocytes the so-called
hepatic-type FAHP is associated with the endoplasmic reticulum and the outer membrane of mitochondria [S]. Interestingly, both FAHP types are
also found inside the nucleus 14, 51. From the abundance of FAHP in tissues with ‘active’lipid metabolism and the ability to bind fatty acids with high
affinity, an involvement o f these proteins in fatty acid
metabolism was inferred. Already in the early days
of FAHP research a variety of studies related to this
field was undertaken even though the proteins were
often not available in pure form and structurally not
characterized. With the advent of detailed structural
information functions were proposed in a more
rationalized manner for the nonenzymic FAHPs, e.g.
roles in signal transduction and in regulation of cell
growth.
Dissecting the structure of cardiac
fatty acid-binding protein
The cI)NA for bovine heart FAHI’ [ h ]codes for a
132 amino acid protein with a high proportion of
P-sheet, as predicted from secondary structure calculations. Plasma desorption Inass spectrometry
indicates a molecular mass of 14675,k 10 Da for
the purified protein 17I in good agreement with that
deduced from the cI)NA, if one takes into account
that the protein is N-terminally blocked by an acetyl
group. The latter finding was corroborated by mass
analysis of N-terminal peptides. In many cases
FAHPs occur as isoforms that differ in their charge;
for example cardiac FAHP from bovine heart has pl
4.9- and pI 5.1-isoforms. A complete protein
Abbreviations used: e.l.i.s.a., enzyme linked inimunosorbent assay; FAHI’. fatty acid-binding protein; MIIGI,
mammary derived growth inhibitor.
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sequence determination o f the puritied isotorms
pin-pointed the molecular basis for this heterogeneity to a single asparagine/aspartate vxchange in
position 98 [ 7 ] .The pI S.1 -isoform with asparagine
in this position was co-linear to the cI )NA (Fig. 1 a )
and the question arose. whether the two charge isoforms are translated from different niKNAs or
result from post-translational deamidation, be it
spontaneous or enzymic. Analysis by isoelectric
focusing of immunoprecipitated [‘isImethioninelabelled products translated in vitro from total
mRNA of bovine heart and from positive hybrid
selected cardiac FAHI’-mRNA on the one hand
showed two proteins that co-migrated with the
authentic pI 4.9- and pI 5.1-isofomis. On the other
hand translation of recombinant mIINA derived
from the cDNA encoding the pI 5.1-isoform produced indeed the pI 5.1-isoform only (N. Hartetzko
& F. Spener, unpublished work). ‘I’he result of these
experiments demonstrates that each isoform is
encoded by its specific mKNA and precludes posttranslational deamidation. Interestingly, only the pI
4.9-isoform is found in the mitochondria1 matrix.
Whether a specific import mechanism is responsible for this phenomenon is currently under investigation.
Tertiary structures have been solved for intestinal-type FAHP [ X I and for myetin 1’2 protein [(I],
the latter also a member of the FAHI’-family. ‘I’he
three dimensional structure of the intestinal FAHI’/
fatty acid complex at 0.12 nm resolution 1x1 reveals
the ligand fatty acid held inside the protein and an
interaction of the fatty acid’s carboxylic group with
Arg“”’.Modification experiments with phenylglyoxal.
an arginine-specific reagent, indicate that in the
case of cardiac FAHP this basic amino acid interacts
with the ligand fatty acid as well (‘1’. Ijiirchers & F.
Spener, unpublished work). Recently, tertiary structures of a further two members from this protein
family were published, that of cardiac FARP from
bovine heart [ l o ] (Fig. 16) and that of murine
adipocyte FAHP [ 111. Common to all structures is a
highly conserved structural motif that consists of
two P-sheets, each built from five antiparallel Pstrands and one being interrupted by two short ahelices. The barrel-like P-sheets provide the
environment for the fatty acid bound in the fatty
acid-binding protein; dissociation constants are in
Lipid-BindingProteins
Fig. I
Structure of the cardiac type FABP
( a ) Primary structures of cardiac FABP from bovine heart and of MDGl from bovine mammary gland. Differences are marked by an
asterisk. (b) Schematic representation of tertiary structure of cardiac FABP. Amino acids of interest that are discussed in the text are
marked by an arrow. P, phosphorylation site; FA, interaction with fatty acids; M, mutagenesis t o convert cardiac FABP t o MDGI. The
shaded /%strand represents the sequence (Thr'21-Gln131)
of the inhibitory peptide.
* *
20
40
AC-~DAFVGTWKLVDSKNFDDYMKSLGVGFATRQVGNMTKPT~
Ac-VDAFVGTWKLVSSENFDDYMKSLGVGFATRQVGNMTKPTL
Tvle80
I
IEVNGDTVIIKTQSTFKNYEISFKLGVEFDETTADDRKV
IISVNGDTVIIKTQSTFKNTEISFKLGVEFDETTADDRKV
100
120
KSIVTLDGGKLVHVQKWNGQETSLVREMVDGKLILTLTHG
KSIVTLDGGKLVQVQKWNGQETSLVREMVDGKLILTLTHG
TAVCTRTYEKQA
cardiac FABP
TAVCTRVYEKQ
MDGl
*
*
the range lo-'' to lo-' \I [12, 131. The functional
aspect of fatty acid binding by this protein will be
treated in the next section.
With regard to structure-function relationships three points are worthwhile mentioning.
Firstly, the a-helices from positions 15 to 35 form
an a-helix/turn/a-helix motif well known for a
class of DNA-binding proteins. Taking into account
that cardiac FABP is present in the nucleus, one
may speculate over a role for this protein in gene
regulation. Work is in progress to study the import
of cardiac FABP and of mutants lacking the ahelix/turn/a-helix motif into the nucleus and their
interaction with DNA. Secondly, a consensus
sequence for tyrosine phosphorylation is present
around Tyr", which may indicate that cardiac
FABP is a target in signal transduction. Thirdly, a
stunning 96% homology of cardiac FABP to a
'mammary derived growth inhibitor' is noticed. The
latter two findings are discussed in more detail in
the last two sections.
Modulation of fatty acid metabolism
Among the functions that have been proposed for
FABP and that we may call functions of the first
generation are: (i) to serve as a pool for solubilized
fatty acids, (ii) to promote the cellular uptake of fatty
acids and their utilization, (iii) to protect enzymes
from detergent effects of fatty acids, (iv) to modulate
enzyme activities and (v) to facilitate targetted transport of fatty acids to specific metabolic pathways
(Fig. 2b). Here we shall discuss the modulation of
acyl-CoA ligase and of enzymes of the P-oxidative
system in bovine heart mitochondria.
The functional aspect of compartmentation of
cardiac FABP in the cytosol and mitochondrial
matrix of myocytes was tested under the following
working hypothesis: under conditions of low carbohydrate supply the heart cell gains 75% of the
energy needed from the oxidation of fatty acids.
Thus upon uptake of fatty acids from the blood into
the cell, they partition into the cytosol for transport
to the outer mitochondrial membrane, where they
are activated by acyl-CoA ligase, a prerequisite for
import of the fatty acid into the mitochondrial
matrix and subsequent degradation via P-oxidation.
In this intracellular path cardiac FARP may interfere in a 2-fold manner: firstly, binding the fatty acid
in the cytosol by FABP promotes a targetted transport and/or modulates the activity of the acyl-CoA
ligase, and secondly, the FABP in the matrix may
modulate fatty acid oxidation. This rationale was
tested experimentally in vitro with intact and broken
mitochondria, respectively (J. Hassink, H. Rude1 &
F. Spener, unpublished work).
Under optimized conditions intact mitochondria were monitored for acyl-CoA ligase
activity with 14C-labelledpalmitic, oleic and arachi-
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Biochemical Society Transactions
Fig. 2
Modulation of enzymic activities by cardiac FABP
(0)Modulation
808
of activation of palmitic acid A,
oleic acid nand arachidonic acid A by mitochondrial acyl CoA-ligase in the
presence of cardiac FABP. Incubation conditions: 27 p~ fatty acid, 0.4 mM MgCI,, 2 mM ATP, 0.5 mM EDTA, I mM KCN, 50 p g
mitochondrial protein in 0.22 ml I50 mM TridHCI (pH 8.0, 37°C). ( b ) Model for the promotion of targetted transport of fatty acids by
cardiac FABP. M, plasma and intracellular membranes, respectively; E, membrane-bound enzyme.
w.N
MM
It
@
M
0
I
I
1
I
20
40
60
80
FABP
1
M
Cardiac FABP (PM]
donic acids as substrates in the absence as well as in
the presence of varying amounts of cardiac FABP.
Unreacted fatty acids were removed by solvent
extraction and the amount of newly synthesized
acyl-CoAs in the aqueous phase was determined by
liquid scintillation counting (Fig. 2a). In the absence
of cardiac FABP the activity of acyl-CoA ligase is
similar for palmitate and arachidonate, V,,, for oleate is approximately 50% higher. In the presence of
increasing amounts of cardiac FABP, on the one
hand a concomitant pronounced decrease of oleoylCoA and arachidonoyl-CoA formation is seen.
Already at 40 ,UM FABP arachidonoyl-CoA synthesis is almost abolished. On the other hand the inhibition of palmitic acid activation is less effective.
Taking into account the dissociation constants for
cardiac FABP (PI 4.9-isoform) in complex with palmitic acid (0.88 ,LAM), oleic acid (0.27 ,MUM) and arachidonic acid (0.24 ,UM) (G. Jagschies & F. Spener,
unpublished work) as determined by the Lipidex
method [14], it is obvious that the high affinity of
cardiac FABP for oleate was overcompensated by
the high specificity of the enzyme for this substrate.
The substrate specificity of the acyl-CoA ligase and
the affinity of cardiac FABP for individual fatty
acids explain the preferred channelling of palmitic
and oleic acid into mitochondria, while arachidonate is retained in the cytosol. As shown in the model
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(Fig. 2b) the modulating effect of cardiac FABP on
acyl-CoA ligase is indirect and based on individual
partition equilibria for fatty acids between the
enzyme-containing mitochondrial membrane and
cardiac FABP. This indirect action of cardiac FABP
corresponds with earlier experiments, where we
could not detect direct interaction of this protein
with mitochondrial membranes [4, 131, contrary to
the situation in liver, where hepatic FABP is associated with the outer membrane of bovine liver
mitochondria [ 5, 131.
The functional significance of mitochondrial
localization of cardiac FABP for the degradation of
acyl-CoAs was studied by incubating broken mitochondria with palmitoyl-CoA and oleoyl-CoA as
substrates, again in the absence and in the presence
of varying amounts of FABP. The assay system is
based on the reduction of dichlorophenolindophenol by acyl-CoA dehydrogenases [ 151 and was
optimized in the presence of KCN to inhibit mitochondria] respiration. With a substrate concentration of 10 ,UM acyl-CoA we observed in the
presence of up to 20 ,UM cardiac FABP a decrease
in the reaction rate of about 50%. When the acylCoA concentration was varied at a concentration of
FABP fixed at 10 ,UM,the rate of oxidation at 5-10
,UM acyl-CoA was lower, but at elevated acyl-CoA
concentrations, up to 30 ,UM,the rate of oxidation
Liptd-Bindi ng Proteins
was higher than that without cardiac FABP in the
assay system. One explanation is that the concentration of 'free' acyl-CoA, which inhibits the acyl-CoA
dehydrogenase is lower in the presence of cardiac
FABP. It is possible that the FABP presented under
physiological conditions to the mitochondria1
matrix modulates the activities of the /?-oxidative
enzymes. Under anoxic conditions, when the concentration of acyl-CoAs in the heart rises, cardiac
FABP may prevent the enzymes from being inhibited.
Phosphorylation of cardiac fatty acidbinding protein
Studies on the phosphorylation of proteins in 3T3I,1 cells revealed the adipocyte FABP as cellular
target of the insulin receptor tyrosine kinase [ 16,
171. Insulin-dependent phosphorylation occurred at
TyrI9, which is part of the well known consensus
sequence for tyrosine kinases, Asn-Phe-Asp-AspTyr, a sequence also present in the first a-helix
domain of cardiac FABP. It was thus conceivable
that cardiac FABP takes part in insulin-dependent
signal transduction, as insulin receptors are located
in the plasma membrane of heart muscle cells as
well. Our experimental model comprised a primary
culture of adult rat heart myocytes treated with
ortho-vanadate and phenylarsineoxide to inhibit
phosphatase action and, concomitantly, to allow for
accumulation of phosphorylated species (S. U.
Nielsen & F. Spener, unpublished work). Myocytes
were isolated via Langendorff perfusion of rat heart
and after incubation with ["P]phosphate in the
presence of insulin, cytosolic proteins were separated by two-dimensional gel electrophoresis and
blotted onto PVDF-membranes. Autoradiography
of blots revealed several 15 kDa proteins among
phosphoproteins, yet only one of these could be
precipitated specifically by affinity purified polyclonal antibodies. As expected, this spot did not
match with either of the two isoforms of rat cardiac
FABP observed after immunostaining, as the addition of the negatively charged phosphate moiety
results in the PI of the phosphoprotein being
slightly more acidic than that of the minor isoform.
The amount of rat cardiac FABP actually phosphorylated was estimated to be clearly below
-0.5% of total cardiac FABP in accordance with
data on the phosphorylated adipocyte FABP, which
indicate a stoichiometry for phosphorylation of
0.2% [ 171. In experiments where insulin was
omitted during incubation with ["P]phosphate,
phosphorylated cardiac FABP was detected in autoradiograms only in minute amounts, if at all. This
-
does not prove but certainly is in support of our
hypothesis that cardiac FABP is an intracellular
target of the insulin receptor tyrosine kinase. Further support came from the identification of the
phosphorylated amino acid. For this purpose rat
cardiac FABP was purified from radioactively
labelled myocytes to near homogeneity in one step
by immunoaffinity chromatography and submitted
to tryptic peptide mapping. T.1.c. analysis together
with radiosequencing of the major phosphopeptide
clearly established Tyr19 as substrate for the kinase.
The low degree of phosphorylation observed
in vivo precludes an effect on the gross flow of fatty
acids, as may be deduced from the recent finding
that phosphorylation of adipocyte FABP virtually
abolishes fatty acid binding [18]. Together with a
10-fold reduction in K , for phosphorylation of lipidated adipocyte FABP compared with the apo
FABP [ 191, an insulin-dependent cycling of fatty
acids from the plasma membrane to the endoplasmic reticulum for eventual utilization was suggested. Considering the 500-fold excess of
unphosphorylated FABP, however, that reportedly
also has the potential to target fatty acids between
membranes [20, 211 this catalytical model appears
rather unlikely. Nevertheless, it is tempting to
speculate that phosphorylation of FABP provides
an early intermediate in the signalling chain from
activated insulin receptor to enzymes of lipid metabolism. A possible role for FABP in this transduction is the sensing of the intracellular fatty acid
concentration. Whether the phosphorylated FABP
interacts with other intermediates of the signalling
chain or directly with some specific regulative elements of genes for enzymes of lipid metabolism
[22] is not known at present.
Inhibition of cell growth
In the course of studies on mammary gland
morphogenesis and the role of regulatory polypeptides controlling growth, differentiation and
regression of the mammary epithelium, a factor was
isolated from bovine lactating mammary gland with
inhibitory action on various normal and transformed mammary epithelial cell lines [23]. This
factor, named mammary derived growth inhibitor
(MDGI), surprisingly shares with cardiac FABP
from bovine heart a very high sequence identity. In
Fig. l(a) the seven amino acids that differ in the
primary structure of both proteins are marked. Due
to its binding capacity for fatty acids [24], its
immunological cross-reactivity, its localization in
2D-gels and its abundance, MDGI may be regarded
as an isoform of cardiac FABP. However, the
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growth inhibitory action is not connected to binding
of ligands, as a synthetic peptide spanning amino
acids Thr"l-Gln121 (shaded in Fig. l b ) also inhibits
growth of Ehrlich ascites mammary tumor cells,
although two orders of magnitude less effectively
than the whole protein [25]. Bovine heart cardiac
FARP has no inhibitory effect in this proliferation
assay despite its similarity to bovine MDGI (Fig. 3).
T o exclude the possibility that the manner of purification affects the inhibitory activity or that minute
impurities of authentic MDGI were responsible for
the biological action, we employed recombinant
proteins in the comparison of MDGI and cardiac
FARP. Efforts to clone a cDNA coding for MDGI
from lactating bovine mammary gland were not
successful, in fact, only a cDNA encoding the PI
5.1-isoform of cardiac FARP was obtained [26].
Thus, by site directed mutagenesis [27], cardiac
FARP was sequentially transformed to MDGI.
From corresponding cDNAs highly productive
expression vectors based on the PET system [28]
were constructed and the respective recombinant
FARP, mutants and eventually the recombinant
MDGI were overexpressed in Escherichiu coli strain
RL2 1 (DE3). Recombinant proteins comprised up
to 40% of cytosolic proteins and were purified to
homogeneity by a combination of cation exchange
Fig. 3
Inhibition of proliferation of Ehrlich ascites tumor
cells by MDGI, recombinant MDGI (rMDGI), cardiac
FABP and mutants
Theonine has been replaced by valine in the mutant FABPVal1 2 '
A further mutant is FABPsrop1 3 2 , where, additionally, the codon
for C-terminal Ala'32has been replaced, in the cDNA, with a
stop codon in accordance with the MDGI structure.
I
""1'
10-10
FABPs are more and more understood at the level
of primary structure: the molecular origin of isoforms, the mechanism of ligand binding and even
tertiary structures are being unravelled. Yet the
physiological function of FABP in vivo still remains
enigmatic and a more diverse role in cellular function than previously appreciated has to be envisaged as discussed in the last two sections.
Progress in our understanding of FARP may arise
from studies of the regulation of the protein at the
genomic level [29], e.g. promotor analysis, and from
transfection experiments either with sense or antisense constructs.
The work carried out in the authors' laboratory was
supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 310/A4, A6). T.B. is a recipient of a
fellowship (Graduiertenforderung) of the State of
Nordrhein-Westfalen.
'MDGI
I
10-9
1
10-8
Protein (M)
Volume 20
Conclusion
FABP
I
100
chromatography and gel filtration (A. Kromminga,
A. G. Lezius, R. Grosse & F. Spener, unpublished
work). The antiproliferative capacity of these
proteins is summarized in Fig. 3. Recombinant
FARP has no effect on growth of Ehrlich ascites
tumor cells, whereas the first mutant, in which
ThrIL7of cardiac FABP is exchanged against
of MDGI inhibits growth of tumor cells by -20%
at a concentration of lo-' M. Both recombinant and
authentic MDGI inhibit cell growth at this concentration by -40%. These data suggest that the Cterminal amino acids of MDGI carry the signal for
the inhibition of proliferation of Ehrlich ascites
tumor cells. Indeed, a short stretch of five amino
acids (Arg-Val-Tyr-Glu-Lys), characteristic for
many growth factors, is present in the active
peptide.
MDGI resembles the adipocyte FARP in as
much as both are expressed in a differentiation
dependent manner. Expression of the proteins is
highest in terminally differentiated tissue. MDGI is
virtually absent in virgin mammary gland, scarce in
pregnant mammary gland but abundant in the
lactating tissue [ 13, 261.
I
I
10-7
lo-*
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Received 20 July 1992
labelling of an 88 kDa adipocyte membrane protein by sulpho-N-succinimidyl
long-chain fatty acids: inhibition of fatty acid transport
Carol1 M. Harmon, Paul Luce and Nada A. Abumrad*
Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville,
Tennessee, U.S.A.
Introduction
Di-isothiocyanodisulphonic acid (DIDS) is a potent
irreversible inhibitor of oleate transport in rat
adipocytes. Gel electrophoresis of plasma membranes isolated from rat adipocytes which had been
Abbreviations used: DIL)S, di-isothiocyanodisulphonic
acid; FA, fatty acid; SSO, sulpho-N-succinimidyl oleate;
SSM. sulpho-N-succininiidyl myristate; SSI’, sulpho-Nsuccinimidyl propionate.
*To whom correspondence should be sent, at the following current address: Dept. of Physiology and Biophysics,
State University of New York at Stony Brook. Stony
Hrook, New York 11794-XOhl.I1.S.A.
incubated with tritiated dihydro DIDS demonstrated a prominent peak of radioactivity associated
with membrane component(s) migrating like a protein of M , 80000 to 90000 implicating it in the
membrane transport of fatty acids (FAs). However,
two concerns limited data interpretation. T h e first
one related to the lack of specificity of the compound which, at the concentrations required to produce inhibition of FA uptake, labelled multiple
membrane proteins. Secondly, since DIDS is a
bifunctional reagent, the labelled proteins could
have represented cross-linked Products of smaller
Ones.
W e have synthesized monofunctional non-
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