Inhibition of mitochondrial UCP1 and UCP3 by purine nucleotides and phosphate

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Highlights

  • UCP3 has a unique PN-binding mechanism, different from UCP1.

  • UCP-PN bond lifetimes correlate with PN-phosphorylation and inhibitory strength.

  • Inorganic phosphate is a new physiologically relevant inhibitor of UCP1 and UCP3.

Abstract

Mitochondrial membrane uncoupling protein 3 (UCP3) is not only expressed in skeletal muscle and heart, but also in brown adipose tissue (BAT) alongside UCP1, which facilitates a proton leak to support non-shivering thermogenesis. In contrast to UCP1, the transport function and molecular mechanism of UCP3 regulation are poorly investigated, although it is generally agreed upon that UCP3, analogous to UCP1, transports protons, is activated by free fatty acids (FFAs) and is inhibited by purine nucleotides (PNs). Because the presence of two similar uncoupling proteins in BAT is surprising, we hypothesized that UCP1 and UCP3 are differently regulated, which may lead to differences in their functions. By combining atomic force microscopy and electrophysiological measurements of recombinant proteins reconstituted in planar bilayer membranes, we compared the level of protein activity with the bond lifetimes between UCPs and PNs. Our data revealed that, in contrast to UCP1, UCP3 can be fully inhibited by all PNs and IC50 increases with a decrease in PN-phosphorylation. Experiments with mutant proteins demonstrated that the conserved arginines in the PN-binding pocket are involved in the inhibition of UCP1 and UCP3 to different extents. Fatty acids compete with all PNs bound to UCP1, but only with ATP bound to UCP3. We identified phosphate as a novel inhibitor of UCP3 and UCP1, which acts independently of PNs. The differences in molecular mechanisms of the inhibition between the highly homologous transporters UCP1 and UCP3 indicate that UCP3 has adapted to fulfill a different role and possibly another transport function in BAT.

Abbreviations

AA
arachidonic acid
AFM
atomic force microscopy
BAT
brown adipose tissue
BSA
albumin from bovine serum
CL
cardiolipin from bovine heart
DOPC
1,2-Dioleoyl-sn-glycero-3-phosphocholine
α-CD
alpha-cyclodextrin
DOPE
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
DTT
dithiothreitol
EGTA
ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid
EPL
E. coli polar lipid extract
FFA
free fatty acid
IMM
inner mitochondrial membrane
MES
2-(N-morpholino) ethanesulfonic acid
PN
purine nucleotide
ROS
reactive oxygen species
TREC
simultaneous topography and recognition imaging by AFM
Tris
tris(hydroxymethyl)amino-methane
UCP
uncoupling proteins

Keywords

Mitochondrial uncoupling proteins
Conductance of planar bilayer membrane
Purine nucleotide binding site
Atomic force microscopy
Molecular mechanism of protein inhibition
Arachidonic acid

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1

Current address: Institute of Life Science, University Catholiqué de Louvain, Louvain-la-Neuve, Belgium.