Selective activation of oxidized PTP1B by the thioredoxin system modulates PDGF-ß receptor tyrosine kinase signaling

The inhibitory reversible oxidation of protein tyrosine phosphatases (PTPs) is an important regulatory mechanism in growth factor signaling. Studies on PTP oxidation have focused on pathways that increase or decrease reactive oxygen species levels and thereby affect PTP oxidation. The processes involved in reactivation of oxidized PTPs remain largely unknown. Here the role of the thioredoxin (Trx) system in reactivation of oxidized PTPs was analyzed using a combination of in vitro and cell-based assays. Cells lacking the major Trx reductase TrxR1 (Txnrd1-/-) displayed increased oxidation of PTP1B, whereas SHP2 oxidation was unchanged. Furthermore, in vivo-oxidized PTP1B was reduced by exogenously added Trx system components, whereas SHP2 oxidation remained unchanged. Trx1 reduced oxidized PTP1B in vitro but failed to reactivate oxidized SHP2. Interestingly, the alternative TrxR1 substrate TRP14 also reactivated oxidized PTP1B, but not SHP2. Txnrd1-depleted cells displayed increased phosphorylation of PDGF-ß receptor, and an enhanced mitogenic response, after PDGF-BB stimulation. The TrxR inhibitor auranofin also increased PDGF-ß receptor phosphorylation. This effect was not observed in cells specifically lacking PTP1B. Together these results demonstrate that the Trx system, including both Trx1 and TRP14, impacts differentially on the oxidation of individual PTPs, with a preference of PTP1B over SHP2 activation. The studies demonstrate a previously unrecognized pathway for selective redox-regulated control of receptor tyrosine kinase signaling

‘Sons of athelings given to the earth’: Infant Mortality within Anglo-Saxon Mortuary Geography

FOR 20 OR MORE YEARS early Anglo-Saxon archaeologists have believed children are underrepresented in the cemetery evidence. They conclude that excavation misses small bones, that previous attitudes to reporting overlook the very young, or that infants and children were buried elsewhere. This is all well and good, but we must be careful of oversimplifying compound social and cultural responses to childhood and infant mortality. Previous approaches have offered methodological quandaries in the face of this under-representation. However, proportionally more infants were placed in large cemeteries and sometimes in specific zones. This trend is statistically significant and is therefore unlikely to result entirely from preservation or excavation problems. Early medieval cemeteries were part of regional mortuary geographies and provided places to stage events that promoted social cohesion across kinship systems extending over tribal territories. This paper argues that patterns in early Anglo-Saxon infant burial were the result of female mobility. Many women probably travelled locally to marry in a union which reinforced existing social networks. For an expectant mother, however, the safest place to give birth was with experience women in her maternal home. Infant identities were affected by personal and legal association with their mother’s parental kindred, so when an infant died in childbirth or months and years later, it was their mother’s identity which dictated burial location. As a result, cemeteries central to tribal identities became places to bury the sons and daughters of a regional tribal aristocracy

A novel persulfide detection method reveals protein persulfide and polysulfide-reducing functions of thioredoxin and glutathione systems

Hydrogen sulfide signaling involves persulfide formation at specific protein Cys residues. However, overcoming current methodological challenges in persulfide detection and elucidation of Cys regeneration mechanisms from persulfides are prerequisites for constructing a bona fide signaling model. We here establish a novel, highly specific protein persulfide detection protocol, ProPerDP, with which we quantify 1.52 ± 0.6 and 11.6 ± 6.9 μg/mg protein steady-state protein persulfide concentrations in human embryonic kidney 293 (HEK293) cells and mouse liver, respectively. Upon treatment with polysulfides, HEK293 and A549 cells exhibited increased protein persulfidation. Deletion of the sulfide-producing cystathionine-γ-lyase or cystathionine-β-synthase enzymes in yeast diminished protein persulfide levels, thereby corroborating their involvement in protein persulfidation processes. We here establish that thioredoxin (Trx) and glutathione (GSH) systems can independently catalyze reductions of inorganic polysulfides and protein persulfides. Increased endogenous persulfide levels and protein persulfidation following polysulfide treatment in thioredoxin reductase-1 (TrxR1) or thioredoxin-related protein of 14 kDa (TRP14) knockdown HEK293 cells indicated that these enzymes constitute a potent regeneration system of Cys residues from persulfides in a cellular context. Furthermore, TrxR1-deficient cells were less viable upon treatment with toxic amounts of polysulfides compared to control cells. Emphasizing the dominant role of cytosolic disulfide reduction systems in maintaining sulfane sulfur homeostasis in vivo, protein persulfide levels were markedly elevated in mouse livers where hepatocytes lack both TrxR1 and glutathione reductase (TR/GR-null). The different persulfide patterns observed in wild-type, GR-null, and TR/GR-null livers suggest distinct roles for the Trx and GSH systems in regulating subsets of protein persulfides and thereby fine-tuning sulfide signaling pathways.USD 1,500 APC fee funded by the EC FP7 Post-Grant Open Access Pilot

Characterization of thioredoxin related protein of 14 kDa and its role in redox signaling [Elektronisk resurs]

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