TRP14 is the rate-limiting enzyme for intracellular cystine reduction and regulates proteome cysteinylation

Abstract

It has remained unknown how cells reduce cystine taken up from the extracellular space, which is a required step for further utilization of cysteine in key processes such as protein or glutathione synthesis. Here, we show that the thioredoxin-related protein of 14 kDa (TRP14, encoded by TXNDC17) is the rate-limiting enzyme for intracellular cystine reduction. When TRP14 is genetically knocked out, cysteine synthesis through the transsulfuration pathway becomes the major source of cysteine in human cells, and knockout of both pathways becomes lethal in C. elegans subjected to proteotoxic stress. TRP14 can also reduce cysteinyl moieties on proteins, rescuing their activities as here shown with cysteinylated peroxiredoxin 2. Txndc17 knockout mice were, surprisingly, protected in an acute pancreatitis model, concomitant with activation of Nrf2-driven antioxidant pathways and upregulation of transsulfuration. We conclude that TRP14 is the evolutionarily conserved enzyme principally responsible for intracellular cystine reduction in C. elegans, mice, and humans.

Keywords: Acute Pancreatitis; Cysteine Homeostasis; Protein Cysteinylation; Proteotoxic Stress; Transsulfuration.

Figure 1. TRP14 is the rate limiting enzyme for cystine reduction in HEK293 cells.

(A) Representative western blot of TRP14, Trx1, and TrxR1 in HEK293 (WT) and TRP14 knockdown (KD) and knockout (KO) cells. (B) Representative fluorescence microscopy image showing BODIPY™ FL L-Cystine reduction as green fluorescence in HEK293 and TRP14 KO cells 15 min after the addition of the labeled cystine. (C) BODIPY™ FL L-Cystine reduction capacity of HEK293 and TRP14 KO measured as an increase of BODIPY™ fluorescence over time (2 h). (D) Representative western blot image showing TRP14 and Trx1 levels in HEK293 and TRP14 KO cells overexpressing wild-type TRP14 ( + TXNDC17 WT) or an active-site mutant version of the enzyme ( + TXNDC17 CysMut). (E) BODIPY™ FL l-Cystine reduction capacity of HEK293 and TRP14 KO cells overexpressing wild-type TRP14 ( + TXNDC17 WT) or an active-site mutant version of the enzyme ( + TXNDC17 CysMut). n = 3–6. Data information: Two-way ANOVA and Tukey’s test for multiple comparisons: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. The indicated “n” corresponds to the number of biological replicates. Data plotted corresponds to the mean (columns) ± standard deviation (error bars). Scale bar: 50 μm. Source data are available online for this figure.

Figure 2. Analysis of the flux through the transsulfuration pathway in HEK293 and TRP14 KO cells using heavy sulfur-containing methionine.

Ratio of total (left), steady-state levels of heavy (middle) and steady-state levels of total (right) cystathionine (CTH) (A), cysteine (B), and glutathione (C) under cystine deprivation conditions (4 µM cystine in the culture medium) in HEK293 (solid lines) and TRP14 KO (dashed lines) cells. n = 4 (left and center panels (A–C), n = 20 (right panel (A–C). In the flux analysis, outliers according to Grubbs’ test were excluded. Data information: Two-way ANOVA and Tukey’s test for multiple comparisons: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. The indicated “n” corresponds to the number of biological replicates. Data plotted corresponds to the mean (columns) ± standard deviation (error bars). Source data are available online for this figure.

Figure 3. TRP14 knockout leads to increased viability and Nrf2 activation upon exposure of HEK293 cells to super-physiological cystine concentrations.

(A) Cell viability of HEK293 and TRP14 knockout cells incubated with 500 µM cystine for 24 h. (B) Representative images of NF-κB (cyan), NRF2 (red), and HIF-1α (yellow) activation in pTRAF-transfected HEK293 and TRP14 KO cells treated with 250 µM cystine for 24 h, with 1 µM auranofin and 10 ng/mL TNF-α  used as positive controls. (C) Histograms corresponding to the quantification of the pTRAF signals. n = 3. Data information: Two-way ANOVA and Tukey’s test for multiple comparisons: ***P < 0.001; ****P < 0.0001. The indicated “n” corresponds to the number of biological replicates. Data plotted corresponds to the mean (columns) ± standard deviation (error bars). Scale bar: 500 μm. Source data are available online for this figure.

Figure 4. Importance of TRP14 and the transsulfuration pathway in C. elegans.

(A) Cystine reduction by the C. elegans TXRXR-1/TRP14 coupled enzyme system (green). (B) Table showing growth and viability of C. elegans mutants. (C) Quantification of cbs-1 and cbs-2 mutants growth. The graph shows the developmental stages of cbs-1 and cbs-2 mutants after 3 days incubation at 20 °C from a synchronized egg-lay. The micrographs are representative images of each genotype at day 3 after synchronized egg-lay. n = 248–1157 (B), n = 385–564 (C). Data information: The indicated “n” corresponds to the number of worms used. Data plotted corresponds to the mean (columns) ±  standard deviation (error bars). Source data are available online for this figure.

Figure 5. Protein cysteinylation in TRP14 knockdown HEK cells and decysteinylating activity of the TRP14 and Trx1 enzymatic system.

(A) Representative western blot image showing increased protein cysteinylation levels in TRP14 knockdown HEK293 (TRP14 KD) cells compared to control (parental HEK cells) when incubated with biotinylated cysteine (Cys-BIO). Loading control in Appendix Fig. S3A. (B) Representative western blot image showing the streptavidin-HRP signal after incubating proteins labeled with Cys-BIO from two different cell lysates with the TRP14 enzymatic system for 15–120 min. Loading control in Appendix Fig. S3B. (C) Schematic representation of cysteine residue modifications detected by mass spectrometry in recombinant human peroxiredoxin 2 (Prx2) incubated with BODIPY™ FL l-Cystine. (D) Representative Fox assay to reduce H₂O₂ with 1 µM cysteinylated Prx2 (Prx2-S-S-Cys) (blue line) in the presence of TRP14 enzymatic system, and upon 10 µM TRP14 addition (red arrow); 1 µM Prx2 was used as a control (green line). (E) Representative Fox assay with 1 µM Prx2 (green line) and 1 µM Prx2-S-S-Cys (blue line) activity to reduce H₂O₂ in the presence of TRP14 enzymatic system; and after addition of 10 µM Trx1 (red arrow). (F) Effect of the presence of typical Trx1 substrates on the rate of the decysteinylation reaction using cysteinylated Prx2 as a substrate: the example of insulin. Representative fluorescence image showing that the addition of insulin into the reaction medium resulted in a decreased rate of decysteinylation by the Trx1 system when compared to cysteinylated Prx2 alone. However, insulin addition did not affect the decysteinylation rate of TRP14 (right). The graphs show the densitometric analysis corresponding to Prx2 cysteinylation levels. Loading control in Appendix Fig. S3E. (G) Effect of hydrogen peroxide on the decysteinylation rate by the Trx1 and TRP14 systems. Representative fluorescence images showing that the addition of 100 μM hydrogen peroxide impaired the decysteinylation reaction by the Trx1 system (10 μM Trx1, 10 nM TrxR1, and 1 mM NADPH), whereas it did not affect the rate of the decysteinylation reaction by the TRP14 system (20 μM TRP14, 50 nM TrxR1, and 1 mM NADPH). The graphs correspond to the densitometric analysis of Prx2 cysteinylation levels. Loading control in Appendix Fig. S3E. n = 4. Source data are available online for this figure.

Figure 6. Proteomic analysis of cysteinylated proteins in TRP14 knockdown cells.

The proteomic analysis consisted of three different experiments in which wild-type cells (Control) and TRP14 knockdown cells (TRP14 KD) were incubated with biotinylated cysteine and then enriched for cysteinylated proteins using a streptavidin column. As a control for the technique, cells incubated with cysteine (no label) were processed in parallel to compensate for any nonspecific binding to the column. Once the proteomic data were retrieved, we worked with those proteins that were identified in both wild-type and TRP14 knockdown cells in the three independent experiments but not in the unlabeled samples. The cysteinylated peptides were identified, and the signal intensity of each detected precursor peptide was compared between groups. After performing a t test, we identified 42 proteins that were significantly more cysteinylated in TRP14 KD cells than in WT cells (P < 0.05). These are the proteins shown in the heatmap, which shows the logarithm of the intensity of the precursor signal for each of the proteins (the darker the blue, the higher the signal). This signal is normalized to the sum of the intensities of all peptides detected in the sample. n = 3. Data information: T test where P < 0.05. The indicated “n” corresponds to the number of biological replicates. Data plotted corresponds to the log of the intensity of the precursor peptide signal (shade of blue) for each experiment (cells).

Figure 7. Protein cysteinylation, and GSSG/GSH, cystine/cysteine, and γ-glutamylcystine/γ-glutamylcysteine redox pairs in pancreas from wild-type and TRP14 knockout mice.

(A) Levels of protein cysteinylation, protein gamma-glutamylcysteinylation, and protein glutationylation (n = 5); (B) GSH and GSSG levels, and GSSG/GSH ratio (n = 7; outliers according to Grubbs’ test were excluded); (C) cysteine and cystine levels, and cystine/cysteine ratio (n = 5); (D) γ-glutamylcysteine and γ-glutamylcystine levels, and γ-glutamylcystine/γ-glutamylcysteine ratio (n = 5), in pancreas from wild-type and TRP14 knockout (TRP14 KO) sham mice (control, CT) and upon acute pancreatitis (AP). *P < 0.05 vs. WT control; **P < 0.01 vs. WT control; ^#P < 0.05 vs. KO control; ^##P < 0.01 vs. KO control; ^§P < 0.05 vs. WT pancreatitis; ^§§P < 0.005 vs. WT pancreatitis. Data information: Two-way ANOVA and Tukey’s test for multiple comparisons: *P < 0.05; **P < 0.01; ***P < 0.001. The indicated “n” corresponds to the number of animals used. Data plotted corresponds to the mean (columns) ± standard deviation (error bars). Source data are available online for this figure.

Figure 8. TRP14 deficiency triggers Nrf2 activation and upregulation of the transsulfuration pathway in the pancreas upon pancreatitis markedly diminishing the inflammatory response.

(A) Methionine levels (n = 4). (B) Homocysteine levels (n = 4). (C) Cystathionine levels (n = 4). (D) Representative western blot for cystathionine β-synthase (CBS), and β-tubulin as loading control (n = 4). (E) Thioredoxin (Txn1) mRNA expression (n = 5). (F) Representative western blot for Trx1, and β-tubulin as loading control (n = 4). (G) Thioredoxin reductase 1 (Txnrd1) mRNA expression (n = 6). (H) NAD(P)H dehydrogenase quinone oxidoreductase 1 (Nqo1) mRNA expression (n = 4). (I) Glutamate-cysteine ligase catalytic subunit (Gclc) mRNA expression (n = 4). (J) Heme-oxygenase 1 (Ho1) mRNA expression (n = 5). (K) Representative western blot of NRF2 cytosolic levels in the pancreas (n = 4). (L) Representative western blot for nuclear NRF2 levels upon acute pancreatitis induction (n = 4). Data information: Two-way ANOVA and Tukey’s test for multiple comparisons: (A–C) *P < 0.05; **P < 0.01; (D, E, G–J) *P < 0.05 vs. WT CT; **P < 0.01 vs. WT CT; ***P < 0.0001 vs. WT CT; ^###P < 0.001 vs. TRP14 KO CT; ^####P < 0.0001 vs. TRP14 KO CT; ^§P < 0.05; ^§§P < 0.01; ^§§§§P < 0.0001. The indicated “n” corresponds to the number of animals used. Data plotted corresponds to the mean (columns) ± standard deviation (error bars). Trx1 and NRF2 were analyzed on the same blots, and hence in (F, K) the tubulin loading control was the same for the pancreatitis samples. Source data are available online for this figure.

Figure 9. TRP14 knockout confers protection against acute pancreatitis in mice, and inhibition of the transsulfuration pathway abrogated the protective effect in TRP14 KO mice.

(A) Representative hematoxylin–eosin staining; (B) tissue edema histological score; (C) inflammatory infiltrate histological score; and (D) Myeloperoxidase (MPO) activity in pancreas from wild-type and TRP14 knockout mice under basal conditions and in cerulein-induced acute pancreatitis. (E) Representative hematoxylin–eosin staining; (F) tissue edema histological score; and (G) inflammatory infiltrate histological score in wild-type and TRP14 knockout mice with pancreatitis with and without AOAA. n = 3–6. Data information: Two-way ANOVA and Tukey’s test for multiple comparisons: (B–D) **P < 0.01 vs. WT CT; ****P < 0.0001 vs. WT CT; ^##P < 0.01 vs. TRP14 KO CT; ^####P < 0.0001 vs. TRP14 KO CT; ^§P < 0.05; ^§§§§P < 0.0001; (F, G) **P < 0^.01; ***P < 0.001. The indicated “n” corresponds to the number of animals used. Data plotted corresponds to the mean (columns) ± standard deviation (error bars). Scale bar: 100 μm. Source data are available online for this figure.

Figure 10. Scheme of major findings in this study.

Here we found that TRP14 reduces intracellular cystine to cysteine, as well as reduces protein cysteinylation motifs, being supported in cells by TrxR1 and NADPH. Cystine is taken up into cells through the xCT cystine/glutamate antiporter (orange). Cysteine can also be synthesized from methionine through the transsulfuration pathway, with methionine entering the cell via several transporter systems (blue). Cysteine is needed for GSH synthesis as well as protein synthesis, with both cystine and cysteine being able to cysteinylate proteins under oxidative stress conditions. Several of the involved enzymes and proteins, including CBS of the transsulfuration pathway, GSH synthesis enzymes, xCT itself, TrxR1, and many more enzymes, are target genes for Nrf2, which is typically activated upon oxidative stress. Here we found that knockout of TRP14 lacks major phenotypes under basal conditions due to the provision of cysteine through transsulfuration, although we could detect increased protein cysteinylation compared to wild-type cells (top two panels). Under conditions of oxidative stress, both wild-type and TRP14 knockout cells activate Nrf2 and thus the transsulfuration pathway as well as the Trx system, and here we show that the overall outcome of TRP14 knockout thus becomes context-dependent. In C. elegans, simultaneous knockout of transsulfuration and TRP14 was non-lethal under basal conditions but lethal under oxidative stress conditions. In the acute pancreatitis model in mice, knockout of TRP14 became protective towards tissue damage in conjunction with a more robust Nrf2 activation than in control animals (lower panels). Created with BioRender.com.

Figure EV1. Schematic representation of the genes and alleles in C. elegans used in this study.

Grey boxes indicate exons encoding the ORF and white boxes represent the UTRs. The molecular lesions are depicted in red.