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The long form of human uncoupling protein-3 (hUCP3L) is highly homologous to thermogenin (UCP1), the uncoupling protein of brown fat mitochondria, but is expressed predominantly in skeletal muscle. Its putative role is to regulate the coupling efficiency of oxidative phosphorylation and thus thermogenesis in skeletal muscle, a major thermogenic tissue in higher mammals. To study the functional relevance of hUCP3L, the protein was expressed in yeast cells under the control of the galactose promoter. Expression of hUCP3L induced a series of phenotype changes in the yeast cells. The cellular growth and the mitochondrial membrane potential were both diminished. The portion of cellular respiration coupled to oxidative phosphorylation decreased from 57% to 11% (P 6 0.001) and the cellular heat production, as measured by direct microcalorimetry, was increased by 33.3 þ 3.2% (P 6 0.001) after induction of UCP3L. These observations demonstrate for the first time the intrinsic thermogenic properties of hUCP3L in intact cells.z 1999 Federation of European Biochemical Societies.

Two splice variants of the human uncoupling protein-3 (UCP3L and UCP3S) are highly expressed in skeletal muscle. The properties of UCP3L and S have been compared to those of UCP1 in a heterologous yeast expression system under the control of the galactose promoter. Both UCP3 isoforms were found to strongly impair the coupling efficiency of respiring cells thus resulting in increased thermogenesis. The uncoupling properties of both UCP3L and S could be clearly demonstrated also in isolated yeast mitochondria both in terms of coupled respiration and in the capacity to polarize the inner membrane in conditions of limited substrate availability. Contrary to what was observed with mitochondria containing UCP1, millimolar GDP and ATP had little if any effect on the uncoupling activity of UCP3. A very marked uncoupling of whole cells and isolated mitochondria was observed at very low expression levels of UCP3S indicating that the short isoform is more active than the long one.z 1999 Federation of European Biochemical Societies.

The uncoupling proteins (UCPs) are thought to uncouple oxidative phosphorylation in the mitochondria and thus generate heat. One of the UCP isoforms, UCP3, is abundantly expressed in skeletal muscle, the major thermogenic tissue in humans. UCP3 has been overexpressed at high levels in yeast systems, where it leads to the uncoupling of cell respiration, suggesting that UCP3 may indeed be capable of dissipating the mitochondrial proton gradient. This effect, however, was recently shown to be a consequence of the high level of expression and incorrect folding of the protein and not to its intrinsic uncoupling activity. In the present study, we investigated the properties of UCP3 overexpressed in a relevant mammalian host system such as the rat myoblast L6 cell line. UCP3 was expressed in relatively low levels (< 1 lgAEmg )1 membrane protein) with the help of an adenovirus vector. Immunofluorescence microscopy of transduced L6 cells showed that UCP3 was expressed in more than 90% of the cells and that its staining pattern was characteristic for mitochondrial localization. The oxygen consumption of L6 cells under nonphosphorylating conditions increased concomitantly with the levels of UCP3 expression. However, uncoupling was associated with an inhibition of the maximal respiratory capacity of mitochondria and was not affected by purine nucleotides and free fatty acids. Moreover, recombinant UCP3 was resistant to Triton X-100 extraction under conditions that fully solubilize membrane bound proteins.Thus, UCP3 can be uniformly overexpressed in the mitochondria of a relevant muscle-derived cell line resulting in the expected increase of mitochondrial uncoupling. However, our data suggest that the protein is present in an incompetent conformation.

BackgroundAltered innate immune responses are observed in HD, with hyper-reactive central and peripheral immune cells producing increased levels of pro-inflammatory cytokine in HD patients. Bone marrow transplantation studies and KMO inhibitor administration have both shown that the immune system is a modifier of HD progression. However, a mechanistic understanding of how this relates to mHTT expression has been lacking.AimCharacterise the abnormal function of peripheral myeloid cells and examine alterations in signalling pathways responsible for those changes.ResultsThe production of pro-inflammatory cytokines in response to LPS was elevated in both monocytes and macrophages from HD patients. Induction of mHTT exon 1 expression in a myeloid cell line showed that the hyper-reactive cytokine production is caused by a cell intrinsic effect of exon 1 mHTT expression. Significant changes in the expression levels of several key molecules in the NFκB pathway, such as IRAK1 and AKT1, were identified using PCR signalling arrays. IκB, the endogenous inhibitor of NFκB activity, was more rapidly degraded following LPS stimulation in HD patients compared to controls, potentially leading to increased NFκB activity and altered gene expression. A direct interaction between HTT and IKKγ was demonstrated by co-immunoprecipitation, suggesting the possibility of a scaffolding function of HTT in the NFκB pathway that may be altered by an expanded polyQ-repeat. Knocking-down total HTT protein levels using GeRP-delivered siRNA in human HD macrophages led to a reduction of cytokine production by LPS-stimulated HD cells.ConclusionsTaken together, these data suggests that the dysfunction of primary myeloid cells from HD patients is directly caused by the expression of mHTT and its effects on the NFκB pathway. Lowering HTT levels in human macrophages can at least partially restore normal cell function, underlining the importance of HTT-lowering as a therapeutic approach.