FRAMING THE ROLE OF RHODANESE-LIKE PROTEINS IN CELL REDOX BALANCE IN TWO BACTERIAL MODEL SYSTEMS
Tesi di Dottorato
Data di Pubblicazione:
2012
Citazione:
FRAMING THE ROLE OF RHODANESE-LIKE PROTEINS IN CELL REDOX BALANCE IN TWO BACTERIAL MODEL SYSTEMS / W. Remelli ; tutor: F. Bonomi ; supervisore: F. Forlani ; coordinatore: F. Bonomi. DIPARTIMENTO DI SCIENZE MOLECOLARI AGROALIMENTARI, 2012 Feb 10. 24. ciclo, Anno Accademico 2011. [10.13130/remelli-william_phd2012-02-10].
Abstract:
A key component of the host’s ability to survive bacterial challenge consist in the innate ability of macrophages to ingest and destroy the invading organism via various mechanisms which the most thoroughly studied is oxidative burst (Hanna et al., 1994). In response pathogens have evolved different approaches to survive the severe oxidative stress generated by the host.
Genome analysis have clustered more than 14000 sequences coding for putative rhodanese like proteins. These sequences contain domains structurally similar to those of the extensively studied bovine rhodanese, and were found in more than 2100 species homogeneously distributed in all life's phyla. Proteins belonging to the rhodanese like superfamily (PFAM accession number: PF00581) are characterized by having more than 140 different architectures of the rhodanese domain, that can be present mostly alone or in tandem with another rhodanese domain, or fused to other functional domains. Although only few amino acid residues are conserved among rhodanese-like proteins, their most distinctive structural feature is the active site configuration, that contains an electronegative residue (generally cysteine) surrounded by positively charged residues. This particular architecture allows rhodanese-like proteins to bear a low pKa catalytic residue that can be the clue to explain their biological activity (Bordo et al 2001).
Although the in vitro reported sulfurtransferase activity for the few characterized rhodanese-like proteins is the transfer of sulfur atom from a sulfur donor (e.g. thiosulfate) to a thiophilic acceptor (e.g. cyanide) (E.C. 2.8.1.x), in the last two decades the scientific community has started to indicate biological roles different from cyanide detoxification for rhodanese like proteins. Proposed roles for rhodanese like proteins can be summarized in two different but complementary fields. Rhodanese-like proteins can function as source of bioactive sulfur equivalents by the formation of a persulfide sulfur on a cysteine residue (R-SSH) (Cartini et al 2011), or can be involved in maintaining redox homeostasis acting as a direct or indirect scavengers of reactive oxygen species (ROS).
My PhD research project was devoted to unravel the biological roles of rhodanese-like proteins using two prokaryotic model systems: the Azotobacter vinelandii and the Bacillus subtilis system.
A. vinelandii is a Gram negative bacterium of the Pseudomonadaceae family in which redox balance must be carefully controlled due to its ability to fix molecular nitrogen via the molybdenum-iron-sulfur cluster enzyme nitrogenase (Setubal et al., 2009). The A. vinelandii genome possesses 14 ORFs coding for rhodanese like proteins with the tandem domain rhodanese-like protein RhdA (Gene ID: 7759697) being responsible for more than 80% of the crude extract thiosulfate:cyanide sulfurtransferase (TST) activity (Cartini et al., 2011). RhdA was widely studied in our lab from both structural and functional point of views.
Starting from the evidence that the rhdA null-mutant strain (MV474) showed altered sensitivity to oxidative events (Cereda et al., 2009), I investigated the nature of the endogenous oxidative stress induced in A. vinelandii by the absence of RhdA. I found that in MV474 strain the ratio GSH/GSSG was misregulated, and the levels of lipid hydroperoxides were significantly increased, although defensive activities against oxidative stress damage were activated (e.g. upregulation of the ahpC gene, coding for Alkylhydroperoxidase C, a member of the OxyR regulon). Furthermore, rhdA expression was highly induced in the A. vinelandii strain (UW136) when the oxidative stress was performed by the incubation with the superoxide generator phenazine methosulfate (PMS). These results
Tipologia IRIS:
Tesi di dottorato
Keywords:
rhodanese-like proteins ; thiyl radical ; redox homeostasis ; glutathione ; hydroxil radical ; bacteria
Elenco autori:
W. Remelli
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