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A chemical probe to monitor the parkinsonismassociated protein DJ-1 in live cells Jonas Drechsel, Franziska A. Mandl, and Stephan A. Sieber ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.8b00633 • Publication Date (Web): 16 Jul 2018 Downloaded from http://pubs.acs.org on July 17, 2018

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ACS Chemical Biology

A chemical probe to monitor the parkinsonism-associated protein DJ-1 in live cells Jonas Drechsel1, Franziska A. Mandl1, Stephan A. Sieber1,* 1

Center for Integrated Protein Science (CIPSM), Technische Universität München,

Department of Chemistry, Lichtenbergstraße 4, 85747 Garching, Germany. * To whom correspondence should be addressed: S.A.S., [email protected]

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Abstract Reactive oxygen species (ROS) play an important role in the onset of Parkinson`s disease (PD) and deciphering protective mechanisms is a major goal for therapeutic development. Here, DJ-1 (PARK7) gained major attention when a conserved cysteine residue with a putative role in oxidative stress sensing/protection was linked to PD. Inspired by previous studies with a bacterial homolog of DJ-1 several aminoepoxycylcohexenones were screened for enzyme inhibition and a chemical probe with specificity for the human ortholog was selected for cellular studies. The probe selectively labeled the cysteine oxidation sensor and whole proteome analysis in HeLa, A549 and SHSY5Y cell lines confirmed strong enrichment of reduced DJ-1 as the most prominent target. Increasing levels of oxidative stress diminished this signal demonstrating the utility of our tool compound for selective in situ monitoring of this important biomarker in its reduced state.


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Introduction Parkinson`s disease (PD) is an age-related, progressive disorder, hallmarked by degeneration of dopamine neurons in the substantia nigra1. Reactive oxygen species (ROS) generated in these cells are believed to be important for the onset of PD, its progression and pathogenesis2,3. DJ-1 (PARK7) gained major attention in PD research due to its putative role in oxidative stress sensing4–8 and protection of the cell against redox stress by a mechanism involving oxidation of a highly conserved cysteine residue (Cys106)1,6. This residue forms four distinct states: the reduced thiol as well as oxidized sulfenic acid, sulfinic acid and sulfonic acid6,11–13. Although it was shown that oxidation to sulfinic acid is mandatory for DJ-1´s cytoprotective function, it is unclear, if this oxidation is directly important for cellular protection by ROS scavenging or connected to regulation of protein function6,14. Moreover, the exact physiological functions of DJ-1 are still enigmatic and additional roles as e.g. cysteine protease, transcriptional activator, redox-regulated chaperone, guanine glycation repair and deglycation enzyme were reported1,9,10. To reveal further insights into its mechanism, oxidation states of DJ-1 were investigated with specific antibodies and confirmed higher levels of oxidized DJ-1 in erythrocytes of PD patients as compared to healthy subjects15. However, a direct readout of the different redox states is still challenging. Recently, chemical probes were introduced to monitor cysteine oxidation directly16. Here, indirect procedures such as labeling thiols using iodoacetamide after specific reduction and methods to directly monitor S-sulfenylation using dimedone and bicyclononyne probes were successfully applied16–21. In addition, sulfinic acid modified proteins including DJ-1 could be detected with aryl-nitroso, S-nitrosothiol and maleimide probes16,22–24. However, these probes monitor global oxidation of cysteine in lysates and lack specificity for DJ-1. Monitoring the oxidation states of DJ1 in living cells directly would be ideal to elucidate the function of this important PD Page 3 ACS Paragon Plus Environment


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biomarker in diverse disease settings. Amino-epoxycyclohexenones (AECH) were recently shown to interfere with bacterial redox enzymes and labeling with a AECH probe revealed high selectivity for sigma cross reacting protein, a close homolog of human DJ-125,26. We therefore set out to investigate the opportunity to use this compound class to monitor DJ-1s expression state.

Results and Discussion A collection of previously established AECHs was tested against recombinant DJ-1 (Figure 1A)25. Although the molecular function and catalytic activity of DJ-1 is not fully elucidated, we were able to employ a reliable assay based on its esterase activity27. Cys106, as part of the esterase dyad, is essential for turnover and can thus be used as a surrogate to measure modification of this crucial oxidation sensor. Based on AECH labeling of the bacterial homolog,25 we anticipated that probe binding occurs also by irreversible opening of the epoxide ring. Out of seven tested AECHs three resulted in strong and four in moderate time-dependent inhibition of DJ-1 (Figure 1B). Binding kinetics (Figure S1) were exemplarily determined for one moderate (FM247) and one strong inhibitor (FM242). While the binding of FM242 to DJ-1 was too fast for determination of kobs/I values, FM247 yielded a kobs/I of 17 s-1M-1. This difference in acylation reactivity was further supported by intact protein mass spectrometry (MS) showing that FM247 exclusively mono-alkylated DJ-1, while FM242 yielded partially double modified protein (Figure S2). Moreover, FM242 labeled a mutant protein bearing an active site Cys106Ala mutant suggesting that this probe scaffold, despite its fast binding kinetics, is not suitable for selective modification of DJ-1 in whole proteomes (Figure S3). In contrast, FM247 did not label the mutant (Figure S3) and was therefore selected as a probe with fine-tuned reactivity for further DJ-1 studies.


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Ideally the AECH scaffold would not only enable specificity for DJ-1, but also provide a direct readout of the thiol oxidation sensor. Hence, we next tested the binding mode of FM247 and a close analog lacking the epoxide ring (FM321). In line with previous results with the bacterial enzyme, only the epoxide containing probe was able to react with the DJ-1’s active site cysteine (Figure 1C). Three equivalents of FM247 were already sufficient to abolish esterase activity (Figure S4). Addition of the probe destabilized the enzyme as shown by a negative thermal shift of 5 °C and 10 °C upon binding of 1 and 10 equivalents of probe, respectively (Figure S5). No such shift was observed in case of the active site Cys106Ala mutant, further corroborating this binding site. Interestingly, oxidation of DJ-1 with H2O2 to the sulfinic acid resulted in enzyme stabilization (Figure S5). This is in line with crystal structure information showing stabilization of oxidized Cys106 via a pronounced H-bond network28. Intact protein MS of oxidized DJ-1 further revealed that FM247 did not modify the sulfinic acid, thereby enabling a direct and specific readout of the reduced state (Figure 1D).



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Figure 1: A) Structures of amino-epoxycyclohexenone probes. B) Probe dependent inhibition of recombinant DJ-1 monitored via esterase assay at 410 nm. Shown are curves from triplicate measurements. C) Intact protein MS measurements of DJ-1 labeled with inhibitor FM247 shows complete conversion of the protein, whereas the inactive probe FM321 did not modify DJ-1. D) Intact protein MS measurements of oxidized DJ-1-SO2H incubated with inhibitor FM247 showed no reaction with the protein.

FM247 is equipped with an alkyne handle suitable for facilitating activity-based protein profiling (ABPP) in live cells29,30. First, we tested the probe binding in E. coli cells expressing DJ-1. Cells were treated with the probe at various concentrations, lysed

and

conjugated

to

rhodamine

azide

via

click

chemistry.

SDS-gel

electrophoresis and subsequent fluorescent scanning revealed a strong protein signal at 23 kDa, visible down to 1 µM probe concentration, that was absent in cells lacking DJ-1 expression (Figure 2A). Furthermore, purified DJ-1 could be clearly labeled with 0.1-0.3 equivalents of FM247, while the oxidized protein as well as the Cys106Ala point mutant escaped detection (Figure 2B, S6, S7).


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After this proof of concept, we performed analysis of the selectivity of the probe in HeLa and A549 cells. In addition, SHSY5Y cells were included as they are often used in the study of parkinsonism associated pathways. An optimal probe concentration of 10 µM was determined via gel-based labeling in A549 cells (Figure S8). Spike-in studies with recombinant DJ-1 in A549 lysate demonstrated strong labeling at 10 µM compound concentration (Figure S8) and a detection limit of 50 nM enzyme (Figure S9). Subsequent target identification was performed via quantitative and gel-free analysis utilizing stable isotope labeling of amino acids in cell culture (SILAC) (Figure 2C). Cells grown in SILAC media were either treated by FM247 or DMSO as control, lysed and conjugated to biotin azide via click chemistry. Biotin labeled proteins were enriched on avidin beads and corresponding peptides were released via tryptic digest for HPLC-MS/MS analysis. Throughout all cell lines DJ-1 clearly showed up as the most prominent hit (Figure 2D, S10). For example, its enrichment in HeLa cells is 170-fold demonstrating a desired specificity for DJ-1 in complex proteomic samples (Figure 2D). Additional, yet lower enriched proteins, which are involved in diverse cellular functions such as cell redox homeostasis and protein folding, were detected as well (Table 1). With this proven selectivity for DJ-1, we next tested, if the probe is also able to detect different oxidation states of the enzyme in situ. For this, HeLa cells were treated with H2O2 at two concentrations and the extent of DJ-1 labeling by FM247 was subsequently monitored via HPLC-MS/MS proteomic analysis and label-free quantification (LFQ) (Figure 2E, S11). Satisfyingly, the enrichment of DJ-1 strongly decreased upon these oxidative conditions by about 60% (Figure 2E).



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Figure 2: A) SDS-gel of DJ-1 labeled with various concentrations of FM247 detected by in-gel fluorescence scanning in the Rosetta2 expression strain. No labeling was observed for the sample without induction of DJ-1 expression (n.i.). Whole gel is shown in Figure S12. B) Labeling of recombinant DJ-1 with FM247 after treating the protein with various concentrations of H2O2, showed the oxidation-state dependent binding of the probe. C) Schematic depiction of a gel-free quantitative proteomic experiment using SILAC. D) Results of the quantitative SILAC experiments using FM247 in the human cancer cell line HeLa. The volcano plot displays the statistical significance of protein enrichment levels as a function of protein enrichment ratios from probe treated to control cells. Cut offs are at a -log10(p-value) of 2 and a t-test difference of 2.5. (indicated by solid lines) E) Decrease of the DJ-1 labeling intensity after in situ pre-incubation with H2O2.

Table 1: The 10 strongest enriched proteins using FM247 in the human cancer cell line HeLa.

Protein names

Significance

Enrichment

-log10(p-value)

log2(FM247/DMSO)

Protein deglycase DJ-1

7.21

7.41

Heme oxygenase 2

4.44

5.57

Protein disulfide-isomerase

6.14

5.47

Hydroxymethylglutaryl-CoA synthase, cytoplasmic

4.98

5.32


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Voltage-dependent anion-selective channel protein 3

7.12

4.72

ERO1-like protein alpha

5.70

4.66

Thymidylate synthase

5.40

4.35

Thioredoxin domain-containing protein 5

7.21

4.28

OCIA domain-containing protein 1

3.48

4.28

Voltage-dependent anion-selective channel protein 2

6.85

4.25

Conclusion DJ-1 gained attention due to its link to PD and oxidative stress. Although the function of the cysteine sensor is still enigmatic, probes that monitor its oxidation state will help to identify its role in disease. Recently, several probes were introduced that bind to the oxidized cysteine of DJ-1. However, these approaches, designed for a global proteome analysis, lack DJ-1 specificity and have been largely applied in cell lysates. Our novel probe enables in situ profiling of DJ-1 and therefore opens new perspectives for selective study of this protein. For example, FM247 could be applied in combination with a global oxidation probe to directly determine ratios of reduced vs. oxidized DJ-1. Such combined studies might be of particular importance for PD biomarker analysis. Furthermore, FM247 and tools derived thereof are promising for imaging of DJ-1 in live cells to obtain direct information of localization and oxidation states. Given the fact that siRNA-induced knock-down of DJ-1 showed promising results in combination with N-(4-hydroxyphenyl)retinamide for the treatment of cervical cancer, a thiol-selective DJ-1 inhibitor might also be of great interest in the field of chemosensitizers31. Our study thus provides starting points for diagnostic applications as well as further medicinal developments.

Conflict of Interest



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The authors declare no conflict of interest.

Associated Content Supporting Information. Applied methods, Figures S1−S12, detailed MS-data evaluation. This material is available free of charge via the internet at http://pubs.acs.org.

Acknowledgment This work was funded by the Deutsche Forschungsgemeinschaft, SFB749 and CIPSM. We thank K. Bäuml, A. Böth, V. Merold, S. Lechner, A. Piller and M. Wolff for help with experiments and technical assistance

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