Transcript acbp-1

Functions of Acyl-CoA Binding Proteins in C. elegans
Ida Coordt
1
Elle ,
Karina Trankjær
2
Simonsen ,
Pernille Kirstine
1
Birck ,
Thuc Timothy
3
Le ,
Nils Joakim
1
Færgeman
1Department
of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark
2Wine Research Center, University of British Columbia, Vancouver, British Columbia, Canada
3Nevada Cancer Institute, Engelstad Research Building R1068, Las Vegas, Nevada,USA
Results
Introduction
Acyl-CoA esters, the metabolically active form of
fatty acids, are important intermediates in both
anabolic and catabolic processes, but have also
been identified as regulators of ion channels,
enzymes, membrane fusion, and gene expression.
Acyl-CoA binding protein (ACBP) is a small,
primarily cytosolic protein, which binds acyl-CoA
esters with high specificity and affinity. Several
studies have indicated that ACBP binds acyl-CoAs
and delivers them to various enzymes for
membrane assembly, vesicle transport, βoxidation, triglyceride storage, and complex lipid
synthesis.
All eukaryotic species express one or more ACBP
paralogues of which some are basal forms and others are
Paralogue #AA
Domain Structure
multi-domain proteins. C. elegans contains seven
ACBP-1 86
(possibly eight) ACBPs, and all of these have mammalian
orthologues of similar domain structure. ACBP-1 is the
ACBP-2/ 385
shortest form in C. elegans and is considered the most
ECH-4
basal form. ACBP-2 is a domain protein containing a CACBP-3 116
terminal enoyl-CoA hydratase/isomerase domain similar
to mammalian peroxisomal enoyl-CoA isomerase, but is
ACBP-4 146
likely expressed in the mitochondria, not in the
ACBP-5 274
peroxisomes. ACBP-3, -4, and -6 are short forms, while
ACBP-5 contains ankyrin repeats, which probably
ACBP-6 115
mediate protein-protein interactions. MAA-1 is a
MAA-1 266
membrane-associated ACBP involved in endosomal
vesicle transport. The recently identified ACBP-7 is not
ACBP
ECH
ANKYRIN
COILED-COIL
TMD included in this study.
Results
To investigate the localization of each of the ACBPs, we
generated translational ACBP::GFP fusions and injected
them into C. elegans to obtain stable lines expressing the
fusion proteins.
A: ACBP-1 is expressed in the intestine; the main fat
storage tissue in C. elegans, indicating a direct function in
triglyceride metabolism.
B: ACBP-2 is expressed in the intestinal cells and
hypodermis; the other fat storage tissue in the worm.
C: ACBP-3 localizes to muscles and hypodermis.
D + E: ACBP-4 is specifically associated with granular
structures in the intestinal cells; likely lipid droplets.
F: ACBP-5 is expressed in the intestine but at very low
levels.
G + H: ACBP-6 localizes to two specific neurons (possibly
CAN and AVG), suggesting a sensory function related to
acyl-CoA metabolism.
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We then investigated lipid storage and
catabolism in ACBP mutants.
Total triglyceride contents were determined by
an enzymatic method, which measures glycerol
levels and therefore is not affected by possible
differences in fatty acid composition. Lack of
either ACBP-1, -2, and -3 results in reduced
triglyceride levels. The acbp-1 mutant displays
the most significant decrease, indicating a role
for ACBP-1 in triglyceride storage in C. elegans.
Surprisingly, the quadruple mutant displays
normal total triglyceride levels.
Asterisks indicate statistical significance;
*: p<0.05; **: p<0.01; ***: p<0.001.
A: N2
B: acbp-1
C: acbp-2
D: acbp-3
E: acbp-4
F: acbp-5
G: acbp-6
H: acbp-1;6;4;3
We have performed CARS (Coherent Anti-Stokes Raman Scattering) microscopy on each of the acbp
deletion mutants in order to examine differences in lipid droplet number and/or morphology.
CARS microscopy relies on contrast derived from molecular vibrations in C-H bonds. This analysis revealed
that acbp-1 contains a reduced number of lipid droplets that are severely enlarged compared to wild-type
droplets, signifying a possible role for ACBP-1 in lipid droplet formation and/or fusion. The quadruple
mutant displays a diffuse CARS signal in the intestine indicating compromised lipid droplet formation.
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We have determined β-oxidation rates by feeding C. elegans tritiated fatty acids and measuring the
formation of tritiated water.
The acbp-1 and -3 deletion mutants display increased β-oxidation of oleic acid revealing a plausible
explanation for their decreased triglyceride storage levels. The acbp-2 mutant displays decreased βoxidation of oleic acid (unsaturated). Conversely, this mutant has increased β-oxidation of palmitic acid
(saturated). The quadruple mutant displays dramatically increased β-oxidation indicating that lack of all
basal ACBPs elicits a starvation-like phenotype.
The distinct expression patterns of C. elegans ACBPs
demonstrate that these proteins probably perform
specific functions, although we also have data indicating
that lack of one form is compensated by up-regulation of
expression of other forms.
Median
lifespan
N2
14
acbp-1
9
acbp-2
12
acbp-3
14
acbp-4
15
acbp-5
14
acbp-6
14
Lifespan assays have been performed in three independent setups. The graph shown is representative of
all data. The acbp-1 mutant has a significantly decreased lifespan compared to N2 (p<0.0001).
The lifespan of the acbp-1;6;4;3 quadruple mutant has been examined in two separate experiments; it is
not different from N2 lifespan.
Conclusions
The results of the current study demonstrate that the ACBP paralogues in C. elegans are expressed in
distinct tissues and have different effects on lipid metabolism. This indicates that each of the ACBPs
performs a specific function, although we also see compensatory mechanisms in several deletion
mutants.
Future Perspectives
Much more work is required to reveal the molecular mechanisms of ACBP functions.
Current efforts include analysis of acyl-CoA compositions by HPLC and lipidomic analysis by mass
spectrometry.
We plan to examine the role of ACBPs during different types of stress.
Investigation of the skin barrier function along with lipidomic analysis have revealed that acbp-3 mutants
are compromised under hypo-osmotic conditions likely due to reduced levels of sphingomyelin containing
very long chain fatty acids. We are currently working on obtaining EM pictures of this mutant and
performing Hoechst stainings to further evaluate skin function.
Reference
Contact: [email protected] or [email protected]
Elle et al. : ”Tissue- and paralogue –specific functions of acyl-CoA binding proteins in lipid metabolism in C.
elegans”. Biochemical Journal, 2011.