5 research outputs found
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
Rutin protects against H<sub>2</sub>O<sub>2</sub>-triggered impaired relaxation of placental arterioles and induces Nrf2-mediated adaptation in Human Umbilical Vein Endothelial Cells exposed to oxidative stress
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<sub>I</sub>-ACP<sub>II</sub>-ACP<sub>III</sub>) within the CurA PKS module.
We have determined the structure of the isolated holo-ACP<sub>I</sub> 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<sub>I</sub>, 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<sub>I</sub> and form a contiguous surface.
Since the CurA Hal accepts substrate only when presented by one of
the ACPs within the ACP<sub>I</sub>-ACP<sub>II</sub>-ACP<sub>III</sub> tridomain, our data provide insight into the specificity of the
chlorination reaction