Characterization of thioredoxin related protein of 14 kDa and its role in redox signaling

Reversible reduction/oxidation (redox) reactions play key roles in cellular signaling pathways. Particularly cysteine residues in proteins can be modified by reactive oxygen-, nitrogen- or sulfur species (ROS, RNS, RSS), thereby altering the functions of the respective proteins. These modifications can be reversed by two major reductive systems in mammalian cells – the thioredoxin (Trx) and glutathione (GSH) systems. Both contain various representatives of the Trx fold family of proteins, among them the name-giving Trxs being the most prominent. In the cytosolic Trx system, electrons are transferred from NADPH to Trx reductase 1 (TrxR1) and subsequently to Trx1, which reduces a multitude of cellular substrates. Thioredoxin-related protein of 14 kDa (TRP14, TXNDC17) is a sparsely characterized, but evolutionarily well-conserved member of the Trx system. The studies comprising this thesis examined TRP14 in several aspects of redox signaling. In Paper I we investigated the inhibition of TrxR1 by noble metal compounds and their effect on cancer cell survival. Inhibition of the Trx system as anti-cancer strategy is thought to attenuate the antioxidant capacity of cancer cells, thereby leading to cell death. We found that gold (Au), platinum (Pt), and palladium (Pd) compounds all inhibited TrxR1 in vitro, but in a cellular context, the inhibition and cytotoxicity were mainly dependent on the ligand substituents and cellular uptake. Furthermore, we found a covalent crosslink between TrxR1 and TRP14 upon treatment of cells with the antitumor agent cisplatin. We concluded that noble metals are potent TrxR1 inhibitors but Pt compounds, especially cisplatin, trigger highly specific cellular effects, including the covalent complex formation. In Paper II we studied the role of the Trx system in reactivation of oxidized protein tyrosine phoshatases (PTPs) in platelet derived growth factor (PDGF) signaling. Using fibroblasts that lacked TrxR1 (Txnrd1 -/-), we found both an increased oxidation of PTP1B and phosphorylation of the PDGF β receptor (PDGF βR). Consequently, we showed that both Trx1 and TRP14, coupled to TrxR1, are able to reduce oxidized PTP1B in vitro. This study demonstrated that the Trx system, including both Trx1 and TRP14, impacts the oxidation of specific PTPs and can thereby modulate PDGF signaling. In Paper III we established TRP14 as an efficient TrxR1-dependent reductase and denitrosylase. Using several low molecular weight disulfide compounds, we found that, dependent on the substrate, TRP14 can be at least as efficient as Trx1. We also suggested TRP14 instead of Trx1 to be a major intracellular cystine reductase, because Trx1 does not reduce cystine once a preferred substrate such as insulin is present. Acting in parallel with Trx1, we also provide evidence of TRP14 being an efficient cellular reductase for nitrosylated proteins and concluded that TRP14 should be considered as an integral part of the Trx system. In Paper IV we developed a novel method for the detection of protein persulfides named Protein Persulfide Detection Protocol, ProPerDP. The formation of persulfide (-SSH) moieties at regulatory cysteine residues is emerging as a major pathway of hydrogen sulfide (H2S) mediated redox signaling. Using ProPerDP we discovered that both the Trx and the GSH system are potent reduction pathways for poly- and persulfides in cells. These studies reinforce the notion that TrxR1-dependent pathways are not only mediated via its wellknown substrate Trx1. We show that TRP14 is yet another cytosolic oxidoreductase with various intracellular functions, including reduction of PTPs, disulfides, nitrosothiols and persulfides. TRP14 is thereby potentially involved in a variety of different redox signaling pathways

Characterization of Molecular Interactions between ACP and Halogenase Domains in the Curacin A Polyketide Synthase

Polyketide synthases (PKSs) and non-ribosomal peptide synthetases (NRPSs) are large multidomain proteins present in microorganisms that produce bioactive compounds. Curacin A is such a bioactive compound with potent anti-proliferative activity. During its biosynthesis the growing substrate is bound covalently to an acyl carrier protein (ACP) that is able to access catalytic sites of neighboring domains for chain elongation and modification. While ACP domains usually occur as monomers, the curacin A cluster codes for a triplet ACP (ACP_I-ACP_II-ACP_III) within the CurA PKS module. We have determined the structure of the isolated holo-ACP_I and show that the ACPs are independent of each other within this tridomain system. In addition, we have determined the structure of the 3-hydroxyl-3-methylglutaryl-loaded holo-ACP_I, which is the substrate for the unique halogenase (Hal) domain embedded within the CurA module. We have identified the interaction surface of both proteins using mutagenesis and MALDI-based identification of product formation. Amino acids affecting product formation are located on helices II and III of ACP_I and form a contiguous surface. Since the CurA Hal accepts substrate only when presented by one of the ACPs within the ACP_I-ACP_II-ACP_III tridomain, our data provide insight into the specificity of the chlorination reaction