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The mitochondrial F1Fo ATP synthase of the parasite Trypanosoma brucei has been previously studied in detail. This unusual enzyme switches direction in functionality during the life cycle of the parasite, acting as an ATP synthase in the insect stages, and as an ATPase to generate mitochondrial membrane potential in the mammalian bloodstream stages. Whereas the trypanosome F1 moiety is relatively highly conserved in structure and composition, the Fo subcomplex and the peripheral stalk have been shown to be more variable. Interestingly, a core subunit of the latter, the normally conserved subunit b, has been resistant to identification by sequence alignment or biochemical methods. Here we identified a 17 kDa mitochondrial protein of the inner membrane, Tb927.8.3070, that is essential for normal growth, efficient oxidative phosphorylation, and membrane potential maintenance. Pulldown experiments and native PAGE analysis indicated that the protein is both associated with the F1Fo ATP synthase, and integral to its assembly. In addition, its knockdown reduced the levels of Fo subunits, but not those of F1, and disturbed the cell cycle. Finally, analysis of structural homology using the HHpred algorithm showed that this protein has structural similarities to Fo subunit b of other species, indicating that this subunit may be a highly diverged form of the elusive subunit b.

Mitochondrial protein import is essential for Trypanosoma brucei across its life cycle and mediated by membrane-embedded heterooligomeric protein complexes, which mainly consist of trypanosomatid-specific subunits. However, trypanosomes contain orthologues of small Tim chaperones that escort hydrophobic proteins across the intermembrane space. Here we have experimentally analyzed three novel trypanosomal small Tim proteins, one of which contains only an incomplete Cx3C motif. RNAi-mediated ablation of TbERV1 shows that their import, as in other organisms, depends on the MIA pathway. Submitochondrial fractionation combined with immunoprecipitation and BN-PAGE reveals two pools of small Tim proteins: a soluble fraction forming 70 kDa complexes, consistent with hexamers and a second fraction that is tightly associated with the single trypanosomal TIM complex. RNAi-mediated ablation of the three proteins leads to a growth arrest and inhibits the formation of the TIM complex. In line with these findings, the changes in the mitochondrial proteome induced by ablation of one small Tim phenocopy the effects observed after ablation of TbTim17. Thus, the trypanosomal small Tims play an unexpected and essential role in the biogenesis of the single TIM complex, which for one of them is not linked to import of TbTim17., Author summary Trypanosoma brucei and its relatives are prominent pathogens causing human and animal diseases, which mainly affect developing countries. The single mitochondrion of trypanosomes is essential across its entire life cycle. Most organellar proteins are imported by hetero-oligomeric protein complexes in the two mitochondrial membranes. Interestingly, the composition of the two import machineries is remarkably different from their corresponding counterparts in other organisms. In contrast, chaperones termed small Tims that escort hydrophobic proteins across the aqueous intermembrane space are conserved in almost all eukaryotes including trypanosomes. Here we show that a fraction of them interact tightly with the inner membrane translocase. Another fraction is present as soluble 70 kDa complexes, which likely consists of hexamers of small Tims without a defined subunit composition. In other eukaryotes these hexamers are usually composed of two alternating small Tims. Moreover, while some small Tims are involved in import of a core subunit of the inner membrane protein translocase, we found one small Tim that directly mediates the assembly of the translocase complex. Knowing which components of the trypanosomal protein import systems are conserved and which ones are not is essential to evaluate whether mitochondrial protein import might be a suitable drug target.

vorgelegt von Wenger Christoph Zsfassungen in dt. und engl. Sprache Graz, Univ., Masterarb., 2013

von: Christoph Wenger Graz, Univ., Dipl.-Arb., 2011

When rotator cuff deficiency is present with gleno-humeral osteoarthritis, the current treatment is a reverse total shoulder arthroplasty. The reverse shoulder prosthesis has inverted articular surfaces: a metallic ball (glenosphere) is fixed on the scapula side, usually by means of a metallic base plate, screwed within the glenoid bone, and a polyethylene cup is inserted within a metallic humeral component. Although the clinical outcome is rather satisfactory, at least for elderly people, the optimal fixation of the base plate remains open. The goal of this project is to test the hypothesis that a new base plate prototype could improve the mechanical stability and the surgical technique, compared to a classical existing one. The ASTM F2028 testing protocol will be used to evaluate the prototypes vs. the classical designs. Measurement will be done on cadaveric scapulae.