Browsing by Person "Papatheodorou, Panagiotis"

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Funktionen charakteristischer Sequenzmotive endogener und toxischer mitochondrialer Proteine
(2006) Papatheodorou, Panagiotis; Rassow, Joachim
In the course of their biogenesis, mitochondria take up nuclear encoded proteins from the cytosol continuously. Protein import at the mitochondrial outer membrane is mediated by TOM proteins and by TIM proteins at the inner membrane, respectively. Now and then, toxical proteins released by pathogenic bacteria to infected tissue can also reach mitochondria. The present dissertation provides new findings on the role of characteristical sequence motifs that can be identified in endogenous and toxical mitochondrial proteins. In an extensive project the importance of sequence motifs from mitochondrial metabolite carrier proteins in their biogenesis and function was investigated in more detail. It could be shown, that the positively charged presequence of the citrate carrier from Rattus norvegicus is not involved in mitochondrial targeting but rather serves as an internal chaperone. A conserved sequence motif, PX(D/E)XX(R/K), the Carrier Signature, which can be found in all mitochondrial carrier proteins, does also not represent a mitochondrial targeting signal, as could be proven by using the dicarboxylate carrier from Saccharomyces cerevisiae as a model protein. Even the translocation across the outer membrane, the insertion into the inner membrane and the following dimerization of the dicarboxylate carrier are processes occuring independently of the Carrier Signature. Instead, it was discovered, that the Carrier Signature is primarily necessary for the function of metabolite carrier proteins in the inner membrane. In another project it could be shown for the Map toxin from enteropathogenic Escherichia coli strains (EPEC), that it is directed to the mitochondrial matrix, mediated by its typical N-terminal presequence and by the TOM and TIM complexes, respectively. The Map toxin leads then to the fragmentation of the mitochondrial network independent of the mitochondrial fission machinery and to the loss of the mitochondrial membrane potential. Moreover, it could be proven, that an internal conserved sequence motif, WXXXE, is essential for cytotoxicity of the Map toxin in the cytosol and for fission of mitochondria. A lysine residue within the WXXXE sequence serves probably as a locus of sumoylation. The investigations show, that mechanisms of intracellular protein transport are not only important for the biogenesis of mitochondria, but can also be relevant for pathological processes.
Synthesis of biologically active Shiga toxins in cell-free systems
(2024) Ramm, Franziska; Kaser, Danny; König, Irina; Fellendorf, Juliane; Wenzel, Dana; Zemella, Anne; Papatheodorou, Panagiotis; Barth, Holger; Schmidt, Herbert
Shiga toxins (Stx) produced by pathogenic bacteria can cause mild to severe diseases in humans. Thus, the analysis of such toxins is of utmost importance. As an AB5 toxin, Stx consist of a catalytic A-subunit acting as a ribosome-inactivating protein (RIP) and a B-pentamer binding domain. In this study we synthesized the subunits and holotoxins from Stx and Stx2a using different cell-free systems, namely an E. coli- and CHO-based cell-free protein synthesis (CFPS) system. The functional activity of the protein toxins was analyzed in two ways. First, activity of the A-subunits was assessed using an in vitro protein inhibition assay. StxA produced in an E. coli cell-free system showed significant RIP activity at concentrations of 0.02 nM, whereas toxins synthesized in a CHO cell-free system revealed significant activity at concentrations of 0.2 nM. Cell-free synthesized StxA2a was compared to StxA2a expressed in E. coli cells. Cell-based StxA2a had to be added at concentrations of 20 to 200 nM to yield a significant RIP activity. Furthermore, holotoxin analysis on cultured HeLa cells using an O-propargyl-puromycin assay showed significant protein translation reduction at concentrations of 10 nM and 5 nM for cell-free synthesized toxins derived from E. coli and CHO systems, respectively. Overall, these results show that Stx can be synthesized using different cell-free systems while remaining functionally active. In addition, we were able to use CFPS to assess the activity of different Stx variants which can further be used for RIPs in general.