Qualitative Differences in Protection of PTP1B Activity by the Reductive Trx1 or TRP14 Enzyme Systems upon Oxidative Challenges with Polysulfides or H2O2 Together with Bicarbonate

Abstract

Protein tyrosine phosphatases (PTPs) can be regulated by several redox-dependent mechanisms and control growth factor-activated receptor tyrosine kinase phosphorylation cascades. Reversible oxidation of PTPs is counteracted by reductive enzymes, including thioredoxin (Trx) and Trx-related protein of 14 kDa (TRP14), keeping PTPs in their reduced active states. Different modes of oxidative inactivation of PTPs concomitant with assessment of activating reduction have been little studied in direct comparative analyses. Determining PTP1B activities, we here compared the potency of inactivation by bicarbonate-assisted oxidation using H₂O₂ with that of polysulfide-mediated inactivation. Inactivation of pure PTP1B was about three times more efficient with polysulfides as compared to the combination of bicarbonate and H₂O₂. Bicarbonate alone had no effect on PTP1B, neither with nor without a combination with polysulfides, thus strengthening the notion that bicarbonate-assisted H₂O₂-mediated inactivation of PTP1B involves formation of peroxymonocarbonate. Furthermore, PTP1B was potently protected from polysulfide-mediated inactivation by either TRP14 or Trx1, in contrast to the inactivation by bicarbonate and H₂O₂. Comparing reductive activation of polysulfide-inactivated PTP1B with that of bicarbonate- and H₂O₂-treated enzyme, we found Trx1 to be more potent in reactivation than TRP14. Altogether we conclude that inactivation of PTP1B by polysulfides displays striking qualitative differences compared to that by H₂O₂ together with bicarbonate, also with regard to maintenance of PTP1B activity by either Trx1 or TRP14.

Keywords: PTP1B; TRP14 and Trx1; TrxR1; bicarbonate; peroxymonocarbonate; polysulfide; redox regulation.

Figure 1

Bicarbonate/H₂O₂- and polysulfide-mediated inactivation of PTP1B compared. Recombinant PTP1B (600 nM) was treated with 30 µM of H₂O₂ (blue curves) or 8 µM polysulfide (red curves) with and without addition of 25 mM HCO₃⁻ and catalase (20 μg/mL), as indicated in the figure. All incubations were done in 20 mM HEPES, 100 mM NaCl buffer pH 7.4 containing 0.1 mM EDTA, 0.05% bovine serum albumin (BSA) and 1 mM sodium azide, with subsequent measurements of PTP activity at the indicated times. PTP1B activity is given in min⁻¹ (mol product formed/mol PTP1B/min). Data points represent means ± SEM (n = 4).

Figure 2

The reductive thioredoxin 1 (Trx1) or Trx-related protein of 14 kDa (TRP14) systems can prevent inactivation of PTP1B by polysulfides but not by the combination of HCO₃⁻ and H₂O₂. (A): PTP1B (600 nM) was treated with 80 µM H₂O₂ (blue curves) or 20 µM polysulfide (red curves), with and without addition of 25 mM HCO₃⁻ and the presence of TRP14 (10 µM), NADPH (300 µM), and TrxR1 (250 nM), as indicated. PTP activity was determined after 5, 15, and 30 min of incubation and data points represent means ± SEM (n = 3). (B): PTP1B was treated as in panel A but using 10 µM Trx1 instead of TRP14. (C): Treatment of PTP1B with polysulfide was performed as in panel A but in the presence of varying concentrations of TRP14 as indicated in the figure.

Figure 3

Reactivation of HCO₃⁻/H₂O₂-inactivated PTP1B by the Trx1 or TRP14 systems. Recombinant PTP1B was completely inactivated using 100 µM H₂O₂ and 25 mM HCO₃⁻. After desalting, the oxidatively inactivated PTP1B was then incubated with 2 mM DTT (green), or 10 µM of either Trx1 (blue) or TRP14 (red) together with TrxR1 (2.5 µM) and NADPH (300 µM), as indicated. PTP1B activity was then determined after incubations of 5, 15, and 60 min.

Figure 4

Reactivation of polysulfide-inactivated PTP1B by the Trx1 or TRP14 systems. Recombinant PTP1B was completely inactivated using incubation with 100 µM polysulfide. After desalting, the oxidatively inactivated PTP1B was then incubated with 2 mM DTT (green) or 10 µM of either Trx1 (blue) or TRP14 (red) together with TrxR1 (2.5 µM) and NADPH (300 µM), as indicated. Reactivated PTP1B activity was then determined after incubations of 5, 15, and 60 min.

Figure 5

Abrogation of polysulfide-dependent inactivation of PTP1B by addition of the TRP14 redox system. PTP1B (600 nM) was incubated together with 80 µM polysulfide and aliquots were taken for activity measurements at 5, 15, and 30 min. Two minutes before each time point, addition of TRP14 (10 µM), NADPH (2 mM), and the TrxR1 (2 µM) was also performed with specific samples, as indicated with arrows and dashed parts of the curves. Data points represent means ± SEM (n = 3).

Figure 6

Schemes for the regulation of PTP1B activity by the Trx1 or TRP14 systems in relation to oxidation by either polysulfides or peroxymonocarbonate. (A) Model scheme summarizing the data and interpretations of this study, showing how PTP1B activity can be regulated by different redox dependent mechanisms through H₂O₂/HCO₃⁻ and the Trx1 or TRP14 systems. The reaction of H₂O₂ together with HCO₃⁻ yields peroxymonocarbonate (HCO₄⁻), which oxidizes PTP1B to forms (“PTP1B_SOX”), including a sulfenylamide [12], that are amenable to reactivation by either Trx1 or TRP14 (green arrows). Polysulfides (“HS_x”, red) are potently reduced by either Trx1 or TRP14, whereby these enzyme systems indirectly prevent polysulfides from inhibiting PTP1B. Once persulfidated PTP1B has been formed (“PTP1B_SSx”), this can mainly be reactivated by Trx1 but not as efficiently with TRP14 (dashed green arrow). The figure in (B) illustrates schematically how the activities of the Trx/TRP14/TrxR1 system together with Peroxiredoxins (Prxs) can maintain PTP1B activity in a cellular context, with Trx1-depdendent Prxs counteracting H₂O₂ accumulation, both Trx1 and TRP14 reducing polysulfides, and both systems being able to reduce and thereby reactivate oxidatively inactivated PTP1B. From our results in the present study, we conclude that polysulfide-mediated inhibition of PTP1B (dashed red arrow) must overcome the very potent direct reduction of polysulfides seen with the Trx1 and TRP14 systems, while H₂O₂ together with HCO₃⁻ yielding peroxymonocarbonate (HCO₄⁻) can inhibit PTP1B also in the presence of active Trx1 and TRP14 systems. Ultimately the extent of PTP1B activity will control growth-factor (GF) signaling, with more inhibited PTP1B allowing for more signaling activity.