Functional characterisation of the ClpP1P2/Clp-ATPase complex of the ADEP producer Streptomyces hawaiiensis NRRL 15010 in vitro and elucidation of the self-resistance mechanism

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dc.contributor.advisor Brötz-Oesterhelt, Heike (Prof. Dr.)
dc.contributor.author Reinhardt, Laura
dc.date.accessioned 2021-07-29T15:20:00Z
dc.date.available 2021-07-29T15:20:00Z
dc.date.issued 2023-06-15
dc.identifier.uri http://hdl.handle.net/10900/117375
dc.identifier.uri http://nbn-resolving.de/urn:nbn:de:bsz:21-dspace-1173754 de_DE
dc.identifier.uri http://dx.doi.org/10.15496/publikation-58750
dc.description.abstract The caseinolytic protease Clp, highly conserved in eubacteria and eukaryotes, degrades misfolded proteins as well as short-lived regulatory factors by targeted proteolysis. The Clp degradation machinery consists of two stacked heptameric rings, forming the secluded degradation chamber and hexameric Clp-ATPases, which regulate proteolysis by recognising, unfolding and translocating substrates into the proteolytic chamber (Yu and Houry, 2007). The proteolytic core ClpP is tightly regulated by its cognate Clp-ATPases, thereby protecting the cells from unspecific, Clp-ATPase-independent protease activity. Acyldepsipeptides (ADEPs), produced by Streptomyces hawaiiensis NRRL 15010 (Thomy et al., 2019), dysregulate ClpP in a dual mode of action. ADEPs bind to the same hydrophobic pockets as the Clp-ATPases on the one hand, inhibiting the physiological functions of ClpP and on the other hand conferring an independent protease activity to the tetradecameric barrel of the peptidase by opening the axial pores and allosterically activating the catalytic triads (Kirstein et al., 2009, Lee et al., 2010, Li et al., 2010, Gersch et al., 2015, Famulla et al., 2016, Pan et al., 2019, Sass et al., 2011, Silber et al., 2020b). In the current PhD project the Streptomyces hawaiiensis housekeeping Clp peptidase was studied and found to consist of a heteromeric ClpP1P2 complex with distinct but complementary functions. The ADEP-sensitive ClpP1 is the main propeptide processor of itself and ClpP2 and confers activity to the mixed ClpP1P2 complex. Contrariwise, the ADEP-insensitive ClpP2 is proteolytically inactive but interacts with the Clp-ATPases ClpX, ClpC1 and ClpC2 for the degradation of the natural substrates ClgR and PopR, as well as the model substrates casein (ß-and fluorogenic α-casein) and GFP-ssrA. In the presence of ADEP1, the Clp-ATPase mediated degradation of natural and model substrates is not abrogated, but even stimulated, revealing a third mechanism of ADEP action by the concomitant binding of ADEP1 and the Clp-ATPases to opposite sides of the ClpP1P2 barrel. Thus, the physiological function of the Streptomyces Clp protease in the presence of ADEP1 is not inhibited. Rather, the data indicate that unspecific protease activity by homomeric ClpP1 and heteromeric ClpP1P2 complexes are the ADEP mode of killing in Streptomyces. Previously, a sixth clpP gene was discovered downstream of the ADEP-biosynthetic gene cluster, designated clpPADEP, which encodes a Clp peptidase that functions as an ADEP resistance factor (Thomy et al., 2019). The current PhD project reveals molecular insights, how a Clp peptidase mediates producer self-protection against ADEP. In vitro substrate degradation experiments in combination with interaction studies revealed a dual mechanism of resistance mediated by ClpPADEP. To protect the cells from unspecific, Clp-ATPase-independent protease activity by ADEP-activated ClpP1 and/or ClpP1P2, ClpPADEP interacts with ClpP1 and forms lower oligomeric complexes (presumably dimers and/or trimers), thereby inhibiting the formation of ClpP1 homo-tetradecamers and ClpP1P2 hetero-tetradecamers. Given that ClpP is essential for viability in Streptomyces (Viala et al., 2000), the physiological function of the housekeeping ClpP1P2 protease has to be replaced. Experiments employing the two ADEP-insensitive Clp peptidases ClpPADEP and ClpP2 in Clp-ATPase mediated substrate degradation experiments illustrated that ClpPADEPP2 in association with ClpX can degrade the natural substrates ClgR and PopR. Additionally, ClpC1PADEPP2 successfully hydrolysed the model substrate ß-casein, demonstrating the formation of active ClpC1/ClpX-PADEPP2 complexes in vitro. Furthermore, the ClpP2 mutant proteins ClpP2S131A and ClpP2hp were utilised in combination with wild-type ClpPADEP for the ClpC1-mediated degradation of ß-casein, analysing the catalytic activity and Clp-ATPase/ClpP interactions of the ClpPADEPP2 complex. Those experiments indicate that ClpPADEP exhibits proteolytic activity in the ClpPADEPP2 complex, thereby functioning as an ADEP-insensitive replacement of the ADEP-sensitive ClpP1, whereas ClpP2 remains the sole interaction partner for the Clp-ATPases. en
dc.language.iso en de_DE
dc.publisher Universität Tübingen de_DE
dc.rights ubt-podno de_DE
dc.rights.uri http://tobias-lib.uni-tuebingen.de/doku/lic_ohne_pod.php?la=de de_DE
dc.rights.uri http://tobias-lib.uni-tuebingen.de/doku/lic_ohne_pod.php?la=en en
dc.subject.ddc 570 de_DE
dc.subject.other ClpP, Streptomyces, ADEP de_DE
dc.subject.other ClpP, Streptomyces, ADEP en
dc.title Functional characterisation of the ClpP1P2/Clp-ATPase complex of the ADEP producer Streptomyces hawaiiensis NRRL 15010 in vitro and elucidation of the self-resistance mechanism en
dc.type PhDThesis de_DE
dcterms.dateAccepted 2021-06-15
utue.publikation.fachbereich Mathematisch-Naturwissenschaftliche Fakultät de_DE
utue.publikation.fakultaet 7 Mathematisch-Naturwissenschaftliche Fakultät de_DE
utue.publikation.noppn yes de_DE

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