Present and future program

Studies of the novel mitochondrial uncoupling proteins UCP2, UCP3, discovered in 1997, and others that have been implicated to participate in fever (UCP2), body weight regulation (dysfunction of UCPs or their regulations might lead to obesity) and adaptive thermogenesis in skeletal muscle (UCP3). Recently, their function in prolongation of cell lifetime has been discussed.

The department employs using biophysical and physical chemistry techniques to study the mechanism of function of uncoupling proteins and their regulations in the level of isolated proteins, or in the level of intact mitochondria and tissue cultures; molecular biology to construct and produce recombinant proteins and their mutants in E. coli and yeast expression systems; and protein biochemistry techniques to isolate separate and study uncoupling proteins in the purest possible state. Completely equipped laboratories for molecular biology, protein biochemistry, bioenergetics and fluorescence spectroscopy are available.

Scientific cooperation on uncoupling proteins takes place with Keith D. Garlid, Oregon Graduate Institute, Portland, Oregon (16 years), USA; Louis A. Tartaglia (who has discovered UCP2), Millennium Pharmaceuticals, Inc., Cambridge, MA, USA (2 years); Peter Pohl, University of Halle, Germany; on plant uncoupling proteins with Anibal E. Vercesi, UNICAMP, Campinas, Brazil (4 years); on uncoupling proteins and phosphate carrier with R. Krämer, University of Cologne, Germany (4 years); and on the use of EPR with W. E. Trommer, University of Kaiserslautern, Germany (13 years).

Applied research of the department involves development of liposomal forms of photosenzitizers for photodynamic therapy of tumors.

Most important results

1. Immunological confirmation of the existence of novel mitochondrial uncoupling proteins UCP2 in various types of mitochondria and UCP3 in skeletalmuscle mitochondria (32 in 1999); confirmation of fatty acid cycling for recombinant UCP2 and UCP3 and proving the UCPn interaction with purine nucleotides.
2. Experimental proofs for the mechanism of fatty acid cycling, e.g. (42,43 in 1997) and pyruvate cycling for the uncoupling protein-1 of brown adipose tissue mitochondria.
3. Revealing the existence of inactive fatty acids, i.e. fatty acids that are unable to flip-flop across the lipid bilayer membrane.
4. Proving the existence and function of plant uncoupling mitochondrial protein (PUMP).
5. Indication of fatty acid cycling for mitochondrial ADP/ATP and phosphate carrier.
6. Revealing new hydrophobic substrates and inhibitors for mitochondrial phosphate carrier and its inhibition by fatty acids as well as fatty acid cycling as a side function of this carrier.
7. Identifying the 108 pS channel in the inner mitochondrial membrane as the inner membrane anion channel and revealing the Ca-sensitive K channel in mitochondria.

Results of applied research

Development of the liposomal form of TPP (meso-tetrakis-phenylporhyrin) that was much more efficient in experimental photodynamic therapy of human melanoma operated to nu/nu mice than the commercially available Fotosan 3.

Detailed description of results

1. Anti-UCP3 antibodies, raised by operation of the recombinant protein (expressed in Escherichia. coli) into the spleen of minipigs, crossreacted with recombinant UCP2 (expressed in E.coli or in yeast), slightly with UCPI, and indicated the presence of UCP2 antigen in isolated mitochondria from rat heart, kidney, and brain, pig lymphocytes, rabbit white fat and hamster brown adipose tissue, and the UCP3 antigen in rabbit skeletal muscle.

Reconstituted, partially purified, E. coli-expressed UCP2 and UCP3 were shown to mediate H+ uniport solely in the presence of fatty acids, most probably by the fatty acid cycling mechanism.

2. Fatty acid interaction with the mitochondrial UCP1 was studied using the unique azidofatty acid derivative with a high specific radioactivity (4 tritium atoms), synthesized by ing. J. Hanuš, a cooperating partner from the Institute of Experimental Botany, Prague. The discovered competition by alkylsulfonates indicated that monovalent unipolar substrates of UCPI share the transport pathways with fatty acids. Other experiments supported the mechanism of fatty acid cycling, when uniport of fatty acids is made possible by the protein and protonated fatty acids flip-flops back across the mebrane .

Since also pyruvate and other monocarboxylates are translocated by UCP1, we confirmed the possibility of pyruvate cycling, when pyruvate is expelled from the mitochondrial matrix via UCP1 and it returns with a proton by electroneutral transport mediated by the mitochondrial pyruvate carrier .

3. The main support for the fatty acid cycling mechanism has been provided by the discovery of socalled inactive fatty acids (Ježek et al. FEBS Lett. 408, 161166, 1997) such as 12hydroxylauric acid that are unable to flipflop across the lipid bilayer. It has been found that they also are unable to induce H+ uniport in proteoliposomes with reconstituted UCP1, PUMP, UCP2 and UCP3, or ADP/ATP and phosphate carrier, they are unable to provide a charge transfer (FA anion uniport) with UCP1 and PUMP as well as they do not inhibit (like other fatty acids) chloride uniport via UCP1. Consequently, with an "inhibited" flip-flop, an inhibited FA-induced H+ uniport is found, which supports the fatty acid cycling mechanism.

4. A cooperating partner, A. E. Vercesi, discovered in 1995 a plant uncoupling mitochondrial protein (PUMP) and P.J., M.Ž. and J.B. have characterized properties of PUMP isolated from potato and tomato mitochondria (P.J., two papers in J. Biol. Chem., two in J.Bionerg.Biomembr.) or recombinant PUMP cloned from Arabidopsis (J.B.). It has been found that PUMP mediates fattyacidactivated H+ uniport, does not translocate chloride and pyruvate, and is inhibited by millimolar purine nucleotide di and triphosphates. PUMP has been immunologically indicated in mitochondria of various fruits of tropic or mild climate origin, including those exhibiting a climacteric respiratory rise (which might be due to PUMP function) and in seeds, roots, stems and flowers of various plants. MALDImass spectroscopy peptide mapping has shown that isolated PUMP is indeed a product of StUCP gene cloned from the potato gene library.

5. The mitochondrial ADP/ATP carrier has been indicated to mediate fatty acid cycling. This was supported mainly by the fact that azidofatty acid stopped to uncouple mitochondria upon photoreaction, while prior to photoreaction uncoupled mitochondria in CATsensitive manner and inhibited the ADP/ATP carrier.

6. Also the mitochondrial phosphate carrier has been shown to mediate fatty acid cycling, whereas its natural physiological transport mode, the phosphate/H+ symport was found to be inhibited by fatty acids, including their azido derivatives. A new transport substrate (methanephosphonate) and new inhibitors (diphosphonates) were also revealed for the phosphate carrier, which also interfered with the fatty acid interaction ( diphosphonates also inhibited FA cycling and prevented photolabeling by azidofatty acids.

7. It has been demonstrated that the 108pS channel in the inner mitochondrial membrane is able to translocate sulfate phosphate and benzenetricarboxylates and is inhibited by the same inhibitors (Cibacron blue, niguldipine, propranol) and exhibits the same pH dependence as the so- called inner membrane anion channel characterized biochemically. Therefore, it has been concluded that the 108 pS channel is responsible for the behaviour of the innermembrane anion channel, involved in the mitochondrial volume homeostasis.

Publications