3. Results
3.4. Investigation of a putative redox switch in TRC40
Yeast Get3 functions, apart from TA-protein targeting, as a redox-regulated chaperone (Voth et al. 2014). Get3 shares features with Hsp33, a bacterial redox-regulated chaperone (Jakob et al. 1999; Kumsta and Jakob 2009), such as a CXC-Xn -CXXC motif that is the key of the redox switch of Hsp33 (Jakob et al. 1999; Voth et al.
2014). Upon oxidation in vitro, Get3 undergoes structural rearrangements that bury the TA-binding groove, release the Zn2+ ion in the dimer interface and turn Get3 into an ATP-independent holdase. This conformational change is reversible once reducing conditions are restored and Zn2+ is present in the medium. Furthermore, the ATPase activity of Get3 is drastically reduced upon oxidation (Voth et al. 2014). Accordingly, Get3 in vivo colocalizes in foci with diverse chaperones under ATP-deprived conditions. Moreover, Get3 colocalizes with aggregates in glucose-deprived conditions (Powis et al. 2013). Almost nothing is known about the redox behavior of TRC40, which shares homology with yeast Get3. Another metazoan homolog plays a role in the sensitivity to oxidative agents like cisplatin and arsenite in C. elegans (Hemmingsson, Nöjd, et al. 2009; Hemmingsson et al. 2010). My aim was to further elucidate the redox behavior of TRC40 in vitro and explore the behavior of TRC40 under oxidative conditions in vivo in human cell lines.
3.4.1. CXC and CXXC are conserved from Get3 to TRC40
In order to investigate whether the CXC-Xn-CXXC motif present in yeast Get3 was conserved in human TRC40, I aligned the two protein sequences. Get3 has seven cysteines in its sequence whereas TRC40 has eight. Five out of the seven are conserved from yeast Get3 to human TRC40 (in yellow). Four of these are present in the CXC-Xn-CXXC motif confirming the conservation of this motif (Fig. 41A). TRC40 has an extra CXC motif (C53-X-C55) close to the N-terminus due to the appearance of a new cysteine (C53) not present in yeast Get3 (C36 from Get3 is aligned to C55 in TRC40). The presence of an extra CXC in TRC40 could increase cysteine reactivity.
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C86 and C317 from yeast Get3 are not conserved in TRC40. Three non-conserved cysteines are present in TRC40 (in red): the aforementioned C53 plus C205 and C268
(Fig. 41A). The conserved domains between yeast Get3 and TRC40 are highlighted in Fig. 6, Fig. 12A.
Figure 41. Cysteine conservation after Get3 and TRC40 alignment. (A) Alignment of TRC40 and Get3 in different species indicating conserved (yellow) and non-conserved (red) cysteines. The sequences were aligned with Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/). The UniProt accession numbers have been provided in parentheses.
3.4.2. Oxidation decreased TRC40 ATPase activity
First, I purified recombinant TRC40 from E. coli. I purified a His-MBP-tagged version of TRC40 and cleaved the tag to obtain an untagged-TRC40 (Fig. 42A).
To test the ATPase activity of purified TRC40 I applied a protocol to ensure control of the redox state. First, TRC40 was subjected to reduction and then to an oxidation step to fully oxidize TRC40. Next, I performed a NADH-coupled ATPase activity assay to elucidate the relative ATPase activity of the oxidized TRC40. Oxidation of TRC40 resulted in a 50% reduction in ATPase activity compared to reduced TRC40 (Fig. 42B).
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Figure 42. TRC40 shows similar in vitro redox behavior as yeast Get3. (A) Coomassie-stained SDS-PAGE gel coming from the purification of His-MBP-TRC40. (B) Effect of oxidation on the ATPase activity of TRC40. The reduced protein treated with 5 mM DTT (TRC40 reduced) was compared to the oxidized one treated with 2mM H2O2 and 50 µM Cu2+ (TRC40 oxidized) at 37°C. ATPase activity is normalized to the reduced state. (C) Redox state of TRC40 cysteines determining how many thiol groups are available before and after oxidation using the Ellman’s assay. At least three to four biological replicates were analyzed. The graphs show the mean and the error bars represent standard error of the mean.
3.4.3. Recombinant TRC40 is not fully reduced after in vitro redox treatment
Figure 42. TRC40 shows similar in vitro redox behavior as yeast Get3. (A) Coomassie
stained-SDS-PAGE gel coming from the purification of His-MBP-TRC40. (B) Effect of oxidation on the ATPase activity of TRC40. The reduced protein treated with 5 mM DTT (TRC40 reduced) was com-pared to the oxidized one treated with 2mM H2O2 and 50 µM Cu2+ (TRC40 oxidized) at 37°C. ATPase activity is normalized to the reduced state. (C) Redox state of TRC40 cysteines determining how many thiol groups are available before and after oxidation using the Elman’s assay. At least three to four biological replicates were analysed. The graphs show the mean and the error bars represent standard error of the mean.
TRC40
No induction Supernatant Flow throughPellet Wash
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MW (kDa) Ni-NT
A Flow-through TEV cleavageTag elutionTRC40 purif
His-MBP-TRC40
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according to the Ellman’s assay, whereas the oxidized form of TRC40 contained one.
Hence, three cysteines changed oxidation status between the two forms (Fig. 42C).
The primary sequence of TRC40 contains eight cysteines (Fig. 41A), but only four of them could be detected in the reduced form after reduction of the protein (Fig. 42C) suggesting that this reduction treatment resulted in a partially oxidized instead of a fully reduced protein.
Figure 43. TRC40 steady-state levels are not altered upon hypoxia. (A) TRC40 levels in conditions of normoxia, short and long hypoxia (94% N2, 5% CO2 and 1% O2). HIF-1 alpha was used as a positive marker for induced-hypoxia. Cellular lysates were analyzed for Western blot for TRC40. (B) Quantification of the TRC40 signal intensities for the different oxygen conditions from the blots performed in (A). (C) Quantification of the HIF-1 alpha signal intensities for the different oxygen conditions from the blots performed in (A). Four biological replicates were analyzed. The graphs show the mean and the error bars represent standard error of the mean.
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Figure 43. TRC40 steady-state levels are not altered upon hypoxia. (A)
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HIF- activation upon hypoxia
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3.4.4. TRC40 steady-state levels remained unaltered upon hypoxia
I set out to assess whether TRC40 was oxidized by the high levels of oxygen under standard cell culture conditions. Therefore, I analyzed HeLa cells that underwent a hypoxic treatment (94% N2, 5% CO2 and 1% O2) for 6 h or 24 h. Western-blot analysis revealed no detectable changes of the TRC40 steady-state protein levels (Fig. 43A, Fig. 43B).