Effect of muramyl peptides on mitochondrial respiration.

Muramyl peptides have been shown to exert several biological activities including potentiation of humoral and cell-mediated immunity and stimulation of natural resistance. The mode of action of muramyl peptides has not been elucidated fully and the immunological activities of some derivatives have been associated with toxic effects, including pyrogenicity and inflammatory reactions. Nevertheless, the impact of muramyl peptides on mitochondrial respiration has never been addressed. In this study, the in vitro effects of muramyl peptides on rat liver mitochondria were examined. Toxic muramyl peptides induced a significant decrease in respiratory control ratio versus non-toxic analogues. These results were confirmed by in vivo studies in mice and were extended to mitochondria isolated from spleens. Our data address, for the first time, the effect of muramyl peptides on mitochondrial bioenergetics. Further studies are required to reveal the mechanism of mitochondrial toxicity in relation to the damaging effects of toxic muramyl peptides.

Coenzyme Q induces GDP-sensitive proton conductance in kidney mitochondria.

Addition of coenzyme Q(10) (CoQ) at low concentration (29 nmol/mg of protein) to kidney but not liver mitochondria resulted in an increase in proton conductance. This uncoupling activity required fatty acid and was completely inhibited by GDP. CoQ activated when it was likely to be reduced but not when it was likely to become oxidized. However, the redox state of endogenous CoQ did not affect mitochondrial proton conductance. Stimulation by CoQ was not inhibited by cyclosporin A, carboxyatractylate, bongkrekate and catalase but could be reversed by superoxide dismutase. We conclude that CoQ acted in mitochondria through production of superoxide, which mediated uncoupling, probably by acting through uncoupling protein 2.

Role of intrahelical arginine residues in functional properties of uncoupling protein (UCP1).

The functional role of the four intrahelical arginines in uncoupling protein (UCP1) from brown adipose tissue were studied in mutants where they were replaced by noncharged residues. Wild-type and mutant UCP1 were expressed in Saccharomyces cerevisiae. As measured in isolated UCP1, nucleotide binding was largely lost in mutants of R83, R182, and R276 occurring in three repeated domains and common to mitochondrial carrier family, whereas mutation of the UCP typical R91 shows normal binding capacity but > 20-fold lower binding affinity and a near loss of pH dependency of binding. In reconstituted UCP1, fatty acid dependent H(+) transport is retained in all four mutants, but inhibition by nucleotide changes according to the binding ability of UCP1. Cl(-) transport is inhibited only by mutations of arginines in the first domain (R83 and R91). Also in isolated mitochondria H(+) transport and respiration with all four mutants is similar to wt, and inhibition by GDP is found only in R91T. The three "regular" arginines are suggested to influence the nucleotide binding site indirectly via a charge network and the "extra" R91 directly via an ion bond with the previously characterised pH sensor E190. The mutants were also used to assess intrahelical control of UCP1. In the yeast cells expressing UCP1, the aerobic growth could be reduced by fatty acid addition only with the nucleotide insensitive mutants. This demonstrates an intracellular control of UCP1 by nucleotides and fatty acids.

Uncoupling proteins: the issues from a biochemist point of view.

The functional characteristics of uncoupling proteins (UCP) are reviewed, with the main focus on the results with isolated and reconstituted proteins. UCP1 from brown adipose tissue, the paradigm of the UCP subfamily, is treated in more detail. The issues addressed are the role and mechanism of fatty acids, the nucleotide binding, the regulation by pH and the identification by mutagenesis of residues involved in these functions. The transport and regulatory functions of UCP2 and 3 are reviewed in comparison to UCP1. The inconsistencies of a proposed nucleotide insensitive H(+) transport by these UCPs as concluded from the expression in yeast and Escherichia coli are elucidated. In both expression system UCP 2 and 3 are not in or cannot be converted to a functionally native state and thus also for these UCPs a nucleotide regulated H (+) transport is postulated.

Uncoupling proteins 2 and 3 are highly active H(+) transporters and highly nucleotide sensitive when activated by coenzyme Q (ubiquinone).

Based on the discovery of coenzyme Q (CoQ) as an obligatory cofactor for H(+) transport by uncoupling protein 1 (UCP1) [Echtay, K. S., Winkler, E. & Klingenberg, M. (2000) Nature (London) 408, 609-613] we show here that UCP2 and UCP3 are also highly active H(+) transporters and require CoQ and fatty acid for H(+) transport, which is inhibited by low concentrations of nucleotides. CoQ is proposed to facilitate injection of H(+) from fatty acid into UCP. Human UCP2 and 3 expressed in Escherichia coli inclusion bodies are solubilized, and by exchange of sarcosyl against digitonin, nucleotide binding as measured with 2'-O-[5-(dimethylamino)naphthalene-1-sulfonyl]-GTP can be restored. After reconstitution into vesicles, Cl(-) but no H(+) are transported. The addition of CoQ initiates H(+) transport in conjunction with fatty acids. This increase is fully sensitive to nucleotides. The rates are as high as with reconstituted UCP1 from mitochondria. Maximum activity is at a molar ratio of 1:300 of CoQ:phospholipid. In UCP2 as in UCP1, ATP is a stronger inhibitor than ADP, but in UCP3 ADP inhibits more strongly than ATP. Thus UCP2 and UCP3 are regulated differently by nucleotides, in line with their different physiological contexts. These results confirm the regulation of UCP2 and UCP3 by the same factors CoQ, fatty acids, and nucleotides as UCP1. They supersede reports that UCP2 and UCP3 may not be H(+) transporters.

Coenzyme Q is an obligatory cofactor for uncoupling protein function.

Uncoupling proteins (UCPs) are thought to be intricately controlled uncouplers that are responsible for the futile dissipation of mitochondrial chemiosmotic gradients, producing heat rather than ATP. They occur in many animal and plant cells and form a subfamily of the mitochondrial carrier family. Physiological uncoupling of oxidative phosphorylation must be strongly regulated to avoid deterioration of the energy supply and cell death, which is caused by toxic uncouplers. However, an H+ transporting uncoupling function is well established only for UCP1 from brown adipose tissue, and the regulation of UCP1 by fatty acids, nucleotides and pH remains controversial. The failure of UCP1 expressed in Escherichia coli inclusion bodies to carry out fatty-acid-dependent H+ transport activity inclusion bodies made us seek a native UCP cofactor. Here we report the identification of coenzyme Q (ubiquinone) as such a cofactor. On addition of CoQ10 to reconstituted UCP1 from inclusion bodies, fatty-acid-dependent H+ transport reached the same rate as with native UCP1. The H+ transport was highly sensitive to purine nucleotides, and activated only by oxidized but not reduced CoQ. H+ transport of native UCP1 correlated with the endogenous CoQ content.

Site-directed mutagenesis identifies residues in uncoupling protein (UCP1) involved in three different functions.

Using site-specific mutagenesis, we have constructed several mutants of uncoupling protein (UCP1) from brown adipose tissue to investigate the function of acidic side chains at positions 27, 167, 209, and 210 in H(+) and Cl(-) transport as well as in nucleotide binding. The H(+) transport activity was measured with mitochondria and with reconstituted vesicles. These mutant UCPs (D27N, D27E, E167Q, D209N, D210N, and D209N + D210N) are expressed at near wt levels in yeast. Their H(+) transport activity in mitochondria correlates well with the reconstituted protein except for D27N (intrahelical), which shows strong inhibition of H(+) transport in the reconstituted system and only 50% decrease of uncoupled respiration in mitochondria. In the double adjacent acidic residues (between helix 4 and helix 5), mutation of D210 and of D209 decreases H(+) transport 80% and only 20%, respectively. These mutants retain full Cl(-) transport activity. The results indicate that D210 participates in H(+) uptake at the cytosolic side and D27 in H(+) translocation through the membrane. Differently, E167Q has lost Cl(-) transport activity but retains the ability to transport H(+). The separate inactivation of H(+) and Cl(-) transport argues against the fatty acid anion transport mechanism of H(+) transport by UCP. The mutation of the double adjacent acidic residues (D209, D210) decreases pH dependency for only nucleoside triphosphate (NTP) but not diphosphate (NDP) binding. The results identify D209 and D210 in accordance with the previous model as those residues which control the location of H214 in the binding pocket, and thus contribute to the pH control of NTP but not of NDP binding.

Structure-function relationship in UCP1.

The function of uncoupling protein (UCP1) as a H+ transporter regulated by nucleotide binding is elucidated. H+ transport requires fatty acids (FA) with relatively wide structural tolerance. The nucleotide binding site is specific for purine nucleotides and tolerates a number of derivatives. The strong pH dependency facilitates regulation of nucleotide binding and thus H+ translocation. The structure-function relationship of UCP1 has been analysed by various probes and by mutagenesis. According to our model, FA are a cofactor in H+ transport, providing H+ shuttling carboxyl groups in the translocation channel. By mutagenesis, additional H+ translocating groups at both sides of the translocation channel were found. Two pH sensors, controlling nucleotide binding, were identified in accordance with earlier postulates deduced from the pH dependence of nucleoside diphosphate (NDP) and nucleoside triphosphate (NTP). A common pH sensor E190 and a specific pH sensor H214 for triphosphates only, control access to the phosphate binding moiety. The three mitochondrial carrier family characteristic intrahelical arginines are essential for nucleotide binding. Mutagenesis of other charged residues reveals their role in structure stabilisation and/or has more generalised effects due to charge relay networks in UCP1.

Regulation of UCP3 by nucleotides is different from regulation of UCP1.

UCP3 is an isoform of UCP1, expressed primarily in skeletal muscle. Functional properties of UCP3 are still largely unknown. Here, we report about the expression of UCP3 and of UCP1 in inclusion bodies of Escherichia coli. On solubilization and reconstitution into proteoliposomes, both UCP3 and UCP1 transport Cl- at rates equal to the reconstituted native UCP1. Cl- transport is inhibited by low concentrations of ATP, ADP, GTP and GDP. However, no H+ transport activity is found possibly due to the lack of a cofactor presents in UCP from mitochondria. The specificity of inhibition by nucleoside tri- and diphosphate is different between UCP1 and UCP3. UCP1 is more sensitive to tri- than diphosphate whereas in UCP3, the gradient is reverse. These results show a new paradigm for the regulation of thermogenesis at various tissues by the ATP/ADP ratio. In brown adipose tissue, the thermogenesis is correlated with a low ATP/ADP whereas in skeletal muscle, non-shivering thermogenesis is active at a high ATP/ADP ratio, i.e. in the resting state.

In the uncoupling protein (UCP-1) His-214 is involved in the regulation of purine nucleoside triphosphate but not diphosphate binding.

The nucleotide binding to uncoupling protein (UCP-1) of brown adipose tissue is regulated by pH. The binding pocket of the nucleotide phosphate moiety has been proposed to be controlled by the protonization of a carboxyl group (pK approximately 4.5) for both nucleoside diphosphates (NDP) and nucleoside triphosphates (NTP) (identified as Glu-190) and of a histidine (pK approximately 7. 2) for NTP only. Here we identify His-214 as a pH sensor specific for NTP binding only. In reconstituted UCP-1 from hamster, DEPC diminishes binding of NTP but not of NDP. It also prevents inhibition of H+ transport by NTP but not by NDP. Hamster UCP-1 expressed in Saccharomyces cerevisiae was mutated to H214N resulting in only moderate change of the binding affinity for NTP (GTP) but a 10-fold affinity decrease with the bulkier substituent in H214W, whereas the affinity for NDP (ADP) was largely unchanged. The steep decrease with pH of the binding affinity for NTP in wild type (from pH 6.0 to 7.5) was much flatter in the mutants. Also, the pH dependence of binding and dissociation rates was diminished in these mutants. The transport of H+ and Cl- was not affected. Thus, His-214 is only involved in nucleotide binding, whereas, as previously shown, His-145 and His-147 are involved only in H+ transport. The results validate the earlier proposal of a histidine regulating the NTP binding in addition to a carboxyl group controlling both NTP and NDP binding. It is proposed that His-214 protrudes into the binding pocket for the gamma-phosphate thus inhibiting NTP binding and that His214H+ is retracted by a background -CO2- group to give way for the gamma-phosphate.

H+ transport by uncoupling protein (UCP-1) is dependent on a histidine pair, absent in UCP-2 and UCP-3.

UCP from brown adipose tissue of hamster (now UCP-1) expressed in Saccharomyces cerevisiae was used to examine the role of a conspicuous histidine pair H145 and H147 which is conserved among UCP-1 from various animals. Single and double mutants were generated by converting H145 and H147 into neutral residues (H145Q and H147N). As measured by fluorescence of dansyl-GTP binding, the level of expression of the mutant UCP was the same as wild-type (wt) in the isolated mitochondria. With the isolated and reconstituted UCP, transport of H+ and Cl- were measured. The fatty acid dependent H+ transport was reduced to about 10% in the single mutant H145Q and H147N and almost abolished in the double mutant, whereas Cl- transport into these vesicles was not affected as compared to wt. The possible involvement of the His pair in nucleotide binding and its pH dependence were examined by determining the KD and the kinetics for [14C]GTP and [14C]ADP binding. There were no marked changes in the affinity as well as in the binding and dissociation rates toward both these nucleotides in the mutant versus wt. Thus, the involvement in nucleotide binding can be excluded. The His pair is localized on the matrix side, probably at the entrance of the H+ translocation channel in UCP-1. It is absent in the recently discovered UCP-2, and therefore, UCP-2 might be predicted not to be a H+ transporter or to use a different mechanism. UCP-3 is deficient only in the equivalent H145 and thus can be predicted to still sustain a reduced H+ transport. The data support our contention that H+-dissociation side chains of UCP-1 are involved in H+ transport in cooperation with fatty acid carboxyl groups.

Mutagenesis of the uncoupling protein of brown adipose tissue. Neutralization Of E190 largely abolishes pH control of nucleotide binding.

For expression in Saccharomyces cerevisiae the cDNA of the uncoupling protein (UCP) of brown adipose tissue from hamster has been isolated and used to transform yeast cells. Optimized expression conditions yielded 2% of mitochondrial protein as UCP. UCP was isolated, avoiding copurification of ADP/ATP carrier and porin. Intrahelical E190, previously suggested to be the pH sensor for nucleotide binding, was neutralized to glutamine by mutagenesis. In binding titrations with [14C]guanosine 5'-triphosphate (GTP) and with fluorescent dansyl-GTP, near equal binding capacity for GTP was measured in wild-type (wt) and E190Q. The KD for GTP binding to UCP from yeast has the same strong pH dependence as the original UCP from hamster. With both [14C]GTP and dansyl-GTP, the KD in wt increased 16-19-fold from pH 6.0 to 7.5, while in E190Q this increase was only 2.5-2.9-fold. As a result, at pH 7.5, both [14C]GTP and dansyl-GTP bind 6-fold tighter to E190Q than to wt. The binding rate of GTP decreased 10-fold from pH 6.0 to 7.5 in wt and only 4-fold in E190Q. Woodward reagent K (WRK) known to interact specifically with E190 [Winkler, E., Wachter, E., and Klingenberg, M. (1997) Biochemistry 36, 148-155] abolished [14C]GTP and dansyl-GTP binding to wt UCP, whereas binding to E190Q was fully resistant to WRK. H+ and Cl- transport activity in reconstituted vesicles were the same with wt and E190Q. At pH 7.5, 5 microM GTP is unable to inhibit H+ and Cl- transport in wt but inhibits in E190Q to maximum level. The different sensitivity toward GTP versus GDP found in wt is absent in E190Q. Thus, the mutation E190Q results in the predicted gain of function in binding and proves the role of the intrahelical E190 as a pH sensor for nucleotide binding but excludes a role in transport.