Oxidation resistance 1 is a novel senolytic target

Summary The selective depletion of senescent cells ( SC s) by small molecules, termed senolytic agents, is a promising therapeutic approach for treating age‐related diseases and chemotherapy‐ and radiotherapy‐induced side effects. Piperlongumine ( PL ) was recently identified as a novel senolytic agent. However, its mechanism of action and molecular targets in SC s was unknown and thus was investigated. Specifically, we used a PL ‐based chemical probe to pull‐down PL ‐binding proteins from live cells and then mass spectrometry‐based proteomic analysis to identify potential molecular targets of PL in SC s. One prominent target was oxidation resistance 1 ( OXR 1), an important antioxidant protein that regulates the expression of a variety of antioxidant enzymes. We found that OXR 1 was upregulated in senescent human WI 38 fibroblasts. PL bound to OXR 1 directly and induced its degradation through the ubiquitin‐proteasome system in an SC ‐specific manner. The knockdown of OXR 1 expression by RNA interference significantly increased the production of reactive oxygen species in SC s in conjunction with the downregulation of antioxidant enzymes such as heme oxygenase 1, glutathione peroxidase 2, and catalase, but these effects were much less significant when OXR 1 was knocked down in non‐ SC s. More importantly, knocking down OXR 1 selectively induced apoptosis in SC s and sensitized the cells to oxidative stress caused by hydrogen peroxide. These findings provide new insights into the mechanism by which SC s are highly resistant to oxidative stress and suggest that OXR 1 is a novel senolytic target that can be further exploited for the development of new senolytic agents.

2.1 Design and synthesis of PL probes The structure of PL features two Michael acceptors (electrophiles), the C2–C3 and C7–C8 olefins (Figure 1 a) (Adams et al., 2012 ). Our previous studies showed that both Michael acceptors were important for the senolytic activity of PL (Wang et al., 2016 ), indicating that PL may act as an irreversible inhibitor through the conjugated addition of the nucleophiles (e.g., cysteine residue) on its target protein(s) to the Michael acceptors. Taking advantage of the covalent interaction between PL and its target protein(s), we designed PL probes that can be “tagged” for protein purification and identification in live cells (Figure 1 a). Our structure–activity relationship studies revealed that modifications on the trimethoxyphenyl group of PL were tolerated, and an affinity tag could be attached to the para‐position of the phenyl ring. We thus synthesized PL probes that contain a terminal alkyne. These probes can be used to enrich target proteins by reacting with the corresponding immobilized azide through a bio‐orthogonal copper‐catalyzed azide–alkyne cycloaddition (Click Chemistry) (Supporting Information Figure S1 ) (Kolb & Sharpless, 2003 ), which allows us to perform mass spectrometry (MS)‐based proteomic analysis to identify the target proteins. Through a cell‐based senolytic activity assay (Figure 1 b), we selected a PL probe (Figure 1 a) with the same senolytic activity as PL to be used in our target‐protein identification study. As a negative control, we designed a structurally related compound in which the C7–C8 double bond of the PL probe is saturated (CTL probe, Figure 1 a); we modified this bond because previous studies suggested that the Michael acceptor at the PL C7–C8 olefin covalently binds target proteins, while the C2–C3 olefin can facilitate target‐protein binding after reacting with glutathione (Adams et al., 2012 ; Raj et al., 2011 ). The CTL probe was used to subtract nonspecific proteins pulled down by our PL probe. As expected, the CTL probe was ~9‐fold less potent than the PL probe and has no selective toxicity against SCs because the senolytic activity of PL depends on the presence of both Michael acceptors (Figure 1 b) (Wang et al., 2016 ). Figure 1 Identification and validation of OXR 1 as a target of PL . (a) The chemical structures of piperlongumine ( PL ), alkyne‐labeled piperlongumine ( PL probe), and alkyne‐labeled negative control probe ( CTL probe). The C2–C3 bonds are circled in blue, and the C7–C8 bonds are circled in red. (b) Viable cell counts of nonsenescent WI ‐38 cells ( NC s), IR ‐induced senescent WI ‐38 cells ( IR ‐ SC ), and replication‐exhausted senescent WI ‐38 cells ( RE ‐ SC ) 72 hr after treatment with PL , the PL probe, or the CTL probe. Data are presented as the mean ± SE of percent cell viability from two independent experiments. (c) Schematic of the competitive pull‐down procedure by “Click” Chemistry. (d) Numbers of PL ‐binding proteins identified in NC s, IR ‐ SC s, and RE ‐ SC s. The list of the proteins is presented in Supporting Information Tables S1 and S2 . (e) IR ‐ SC lysate was incubated with biotin‐labeled PL in the absence or presence of a fivefold excess of unlabeled PL followed by precipitation with streptavidin‐conjugated agarose beads and immunoblotting of the proteins released from the beads with anti‐ OXR 1 antibodies. An immunoblot of β‐actin in IR ‐ SC lysates was included as an input control for the pull down. (f) Human recombinant OXR 1 protein was incubated with biotin‐labeled PL in the absence or presence of a fivefold excess of unlabeled PL followed by immunoblotting with antibodies against biotin to detect OXR 1 bound to the biotin‐labeled PL probe. An immunoblot of OXR 1 from each reaction was included as an input control

To further confirm that OXR1 is a senolytic target of PL, we examined the effect of PL on OXR1 expression in NCs and IR‐SCs. We found that IR induced a time‐dependent increase in OXR1 mRNA and OXR1 protein in WI‐38 cells, which correlated closely with the time‐dependent induction of senescence as determined by senescence‐associated β‐galactosidase staining (Figure 2 a–c). PL treatment had no significant effect on the levels of OXR1 mRNA and protein in NCs (Figure 2 d, e). In contrast, PL substantially reduced the levels of OXR1 protein but had no effect on OXR1 mRNA expression in IR‐SCs, suggesting that PL regulates OXR1 in IR‐SCs post‐transcriptionally, possibly by inducing its degradation. Indeed, the PL‐induced downregulation of OXR1 was attenuated by suppressing the activity of the proteasome with MG‐132 (Figure 2 f). Moreover, we found that PL selectively increased the levels of poly‐ubiquitylated proteins in IR‐SCs, but not in NCs (Figure 2 g); and IR‐SCs treated with PL had higher levels of both mono‐ubiquitinated OXR1 and poly‐ubiquitylated OXR1 than those treated with vehicle (Figure 2 h). Collectively, these results suggest that PL can selectively reduce the levels of OXR1 in SCs, in part by inducing the proteasomal degradation of OXR1. Figure 2 PL selectively decreases the level of OXR 1 in SC s, in part by inducing OXR 1 proteasomal degradation. (a) Percentage of SA ‐β‐gal–positive cells at different days after IR . (b) The levels of OXR 1 mRNA in WI ‐38 cells at different days after IR are presented as a fold change from un‐irradiated control cells. (c) The levels of OXR 1 protein in WI ‐38 cells at different days after IR . Left panel, representative immunoblot of OXR 1 and β‐actin; right panel, fold changes in OXR 1 expression in irradiated cells relative to un‐irradiated cells after normalization to β‐actin. (d) The levels of OXR 1 mRNA in NC s and IR ‐ SC s after incubation with vehicle (Veh) or PL (5 μM) for 6 hr. (e) The levels of OXR 1 protein in NC s and IR ‐ SC s after incubation with vehicle (Veh) or PL (5 μM) for 6 hr. Left panel, representative immunoblot of OXR 1 and β‐actin; right panel, fold changes in OXR 1 expression in cells relative to vehicle‐treated NC s after normalization to β‐actin. (f) The levels of OXR 1 were determined in NC s and IR ‐ SC s after incubation with vehicle (Veh) or PL (5 μM) for 6 hr in the presence or absence of 5 μM MG ‐132 ( MG ). (g) The levels of poly‐ubiquitylated (Poly‐Ub) proteins in NC s and IR ‐ SC s after incubation with vehicle (Veh) or PL (5 μM) for 6 hr; β‐actin was included as a loading control. (h) The levels of poly‐ubiquitylated (Poly‐Ub) OXR 1 in IR ‐ SC s after incubation with vehicle (Veh) or PL (5 μM) for 6 hr; the cell lysates were incubated with OXR 1 or IgG control and resin for overnight. After extensive washing, ubiquitinated OXR 1 was eluted by SDS sample buffer and immunoblotted with ubiquitin and OXR 1 antibodies. The input cell lysates were also immunoblotted with antibodies to OXR 1 or β‐actin as indicated. ns indicates the nonspecific band. Data in bar graphs are the mean ± SE of two to three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. un‐irradiated cells by one‐way ANOVA . a p < 0.05 vs. NC s treated with Veh; b p < 0.05 vs. IR ‐ SC s treated with Veh by two‐way ANOVA

OXR1 is an important antioxidant protein that protects cells from oxidative stress by regulating the expression of enzymes that detoxify ROS, such as glutathione peroxidase 2 (GTX2), heme oxygenase 1 (HO‐1), and catalase (CAT) (Jaramillo‐Gutierrez et al., 2010 ; Oliver et al., 2011 ; Yang et al., 2015 ). SCs are known to produce high levels of ROS, but they are highly resistant to oxidative stress (Chandrasekaran et al., 2017 ; Davalli et al., 2016 ; Lee et al., 1999 ; Lu & Finkel, 2008 ). This resistance may be due to the increased expression of OXR1 and its downstream targets in SCs; therefore, OXR1 may be a genuine senolytic target. To test this hypothesis, we used two different OXR1 shRNAs (shOXR1‐1 and shOXR1‐2) to knock down the expression of OXR1 in both NCs and IR‐SCs (Figure 4 a). We found that shOXR1‐1 was slightly more effective than shOXR1‐2 at knocking down OXR1 in both cell types; thus, we used shOXR1‐1 (referred to as shOXR1) for the rest of the study. Figure 4 Knocking down OXR 1 selectively kills IR ‐ SC s by downregulating the expression of antioxidant enzymes to sensitize the cells to oxidative stress. (a) The levels of OXR 1 mRNA (top) and OXR 1 protein (bottom) in NC s and IR ‐ SC s transfected with either scramble control sh RNA (shCtrl) or two different sh RNA constructs targeting OXR 1 (sh OXR 1‐1 or sh OXR 1‐2). (b) Expression of glutathione peroxidase 2 ( GPX 2 ), heme oxygenase 1 ( HO ‐1 ), catalase ( CAT ), superoxide dismutase 1 ( SOD 1 ), and SOD 2 mRNA in NC s and IR ‐ SC s transfected with shCtrl or sh OXR 1. Data in (a) and (b) are the mean ± SE of fold changes in mRNA expression compared with shCtrl‐transfected cells from two to three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. shCtrl by unpaired t test. (c) Percent viability of NC s and IR ‐ SC s transfected with shCtrl or sh OXR 1 presented as the mean ± SE of two independent experiments. * p < 0.05 vs. IR ‐ SC s transfected with shCtrl by unpaired t test. (d) Levels of intracellular ROS in NC s and IR ‐ SC s transfected with shCtrl or sh OXR 1 in the presence or absence of 2 mM N ‐acetyl‐ l ‐cysteine ( NAC ). Data are presented as the mean ± SE of mean fluorescent intensity ( MFI ) of dihydrorhodamine 123 ( DHR ) from two independent experiments. * p < 0.05 vs. IR ‐ SC s transfected with sh OXR 1 without NAC by unpaired t test. (e) Percent apoptotic cells in NC s and IR ‐ SC s transfected with shCtrl or sh OXR 1 in the presence or absence of 2 mM NAC are presented as the mean ± SE of two independent experiments. * p < 0.05 vs. IR ‐ SC s transfected with sh OXR 1 without NAC by unpaired t test. (f) Percent viable cells in NC s and IR ‐ SC s transfected with either shCtrl or sh OXR 1 after incubation with 100 μM H 2 O 2 for 3 day. Data are presented as the mean ± SE of percent viable cells compared with their respective controls without H 2 O 2 treatment from two independent experiments. * p < 0.05 vs. IR ‐ SC s transfected with shCtrl by unpaired t test. (g) Percent apoptotic cells in NS and IR ‐ SC s transfected with shCtrl or sh OXR 1 after incubation with 100 μM H 2 O 2 in the presence or absence of 2 mM NAC . Data are presented as the mean ± SE of two to three independent experiments. * p < 0.05 vs. IR ‐ SC s transfected with sh OXR 1 with H 2 O 2 alone by unpaired t test As expected, IR‐SCs expressed significantly higher levels of the known downstream targets of OXR1, that is, GTX2 and HO‐1 and CAT , and other antioxidant enzymes such as superoxide dismutase 1 ( SOD‐1 ) and SOD2 than NCs (Figure 4 b). Knocking down OXR1 slightly reduced the expression of HO‐1 and CAT mRNA but not GTX2 mRNA, in NCs. In contrast, IR‐SCs showed a greater reduction in GTX2 , HO‐1, and CAT mRNA expression after OXR1 knockdown. Knocking down OXR1 also moderately reduced the expression of SOD‐1 and SOD‐2 mRNA in NCs and SOD‐1 mRNA in IR‐SCs. Importantly, knocking down OXR1 significantly reduced the viability of IR‐SCs, but not NCs (Figure 4 c). These findings suggest that OXR1 may be a novel senolytic target. To determine whether OXR1 knockdown selectively killed IR‐SCs through oxidative stress, we measured ROS production and apoptosis in IR‐SCs and NCs after transfection with control shRNA (shCtrl) or shOXR1 in the presence or absence of the antioxidant N ‐acetyl‐cysteine (NAC). The IR‐SCs produced significantly higher levels of ROS than NCs, and IR‐SCs produced even more ROS after knockdown of OXR1 (Figure 4 d). This increase in ROS was associated with a significant increase in apoptosis in IR‐SCs after knocking down OXR1 (Figure 4 e). Importantly, the increases in ROS and apoptosis in IR‐SCs induced by shOXR1 were abrogated by NAC. In contrast, knocking down OXR1 in NCs only slightly increased ROS production, but this had no significant effect on NC apoptosis. These results suggest that knocking down OXR1 selectively kills IR‐SCs by sensitizing them to oxidative stress and inducing

Resistance to apoptosis and oxidative stress is a hallmark of SCs (Childs, Baker, Kirkland, Campisi & Van Deursen, 2014 ; Childs et al., 2017 ) that can be exploited to develop senolytic agents that selectively kill SCs, assuming that the molecules that mediate the resistance can be identified. Indeed, we and others have recently discovered that the upregulation of the antiapoptotic Bcl‐2 family proteins is primarily responsible for the resistance of SCs to apoptosis, and Bcl‐2/xl/w inhibitors such as ABT‐263 are potent senolytic agents (Chang et al., 2016 ; Yosef et al., 2016 ; Zhu et al., 2016 ). Because long‐term treatment with a senolytic drug may be required to prevent/treat age‐related diseases and extend lifespan, there is a concern that Bcl‐2/xl/w inhibitors might be not safe for humans because of their known on‐target (thrombocytopenia) and off‐target toxicities (Roberts et al., 2011 ; Rudin et al., 2012 ; Vogler et al., 2011 ). Thus, it is important that we continue searching for novel senolytic targets to develop safer senolytic agents. In the present study, we used an MS‐based proteomic approach to identify the direct protein targets of PL in SCs, hoping to find new senolytic targets that can be exploited for the development of a safer senolytic drug because PL is a relatively nontoxic, natural product (Adams et al., 2012 ; Raj et al., 2011 ; Wang et al., 2016 ). We designed a PL‐derived probe with senolytic properties similar to PL that can be tagged to resins via a bio‐orthogonal Click Reaction, allowing us to isolate proteins that interact covalently with the probe. Because it can be used with live cells, this approach allowed us to identify more biologically relevant targets than a traditional cell lysate‐based approach with a biotin‐labeled probe, which is not cell permeable and is not active in live cells as a senolytic agent (data not shown). With this approach, we identified 172 potential senolytic targets of PL. These targets have no overlap with known PL‐target proteins that were identified in cancer cell lysates with a biotin‐labeled PL probe (Raj et al., 2011 ), suggesting that PL may have different targets in SCs and cancer cells. Among these possible senolytic targets of PL, we pursued OXR1 because it helps protect cells from oxidative stress by regulating several ROS‐detoxifying enzymes (Jaramillo‐Gutierrez et al., 2010 ; Oliver et al., 2011 ; Yang et al., 2015 ). Our studies showed that OXR1 is upregulated in both prematurely SCs induced by IR and replicative SCs in conjunction with the increased production of ROS and elevated expression of OXR1‐regulated ROS‐detoxifying enzymes such as GPX2, HO‐1, and CAT. In SCs, PL binds OXR1 directly and induces its degradation via the ubiquitin‐proteasome system; this results, in part, from a differential ability of SCs and NCs to take up PL. Genetic knockdown of OXR1 resulted in the selective killing of SCs by disarming their defense against oxidative stress, because the induction of SC apoptosis by knocking down OXR1 was associated with downregulation of GPX2, HO‐1, and CAT mRNA and increased production of ROS, and this was prevented by the antioxidant NAC. These findings and our observations that PL induces the proteasomal degradation of OXR1 and apoptosis of SCs suggest that OXR1 is a senolytic target that has the potential to be exploited for the development of novel senolytic agents, including PL analogs. This study identifies OXR1 as a potential target of PL to mediate PL senolytic activity. However, PL can bind hundreds of proteins in SCs. It has yet to be determined whether other PL‐binding proteins also play a role in mediating the effect of PL on SCs. In addition, we observed that PL increased the levels of polyubiquitinated proteins in IR‐SCs (Figure 2 g), suggesting that PL may also induce dysregulation of the ubiquitin‐proteasomal system and proteostasis in SCs. Whether the dysregulation induced by PL contributes to PL‐induced SC, apoptosis has yet to be investigated.

Cell viability was measured by flow cytometry, as previously described (Chang et al., 2016 ; Wang et al., 2016 ). Dose–response curves were generated, and the half‐maximal effective concentrations (EC 50 values) were calculated with GraphPad Prism 6 software.

The pull‐down experiments were carried out using the Click‐&‐Go™Protein Enrichment Kit (Product No. 1039, Bioconjugate Technology Company, Scottsdale, AZ) by following the protocol from the manufacturer. Briefly, nonsenescent WI38 cells (NCs), IR‐induced senescent WI38 cells (IR‐SCs), and replicative senescent WI38 cells (RE‐SCs) were plated on 10‐cm Petri dishes. The cells were incubated with PL probe or CTL probe at 5 μM for 5 hr, harvested, and lysed in a lysis buffer as described in the protocol. The concentration of the total protein was measured by NanoDrop. The same amount of total protein (4.48 mg in 800 μl) from each cell type was incubated with azide‐agarose resin in the presence of a copper catalyst solution overnight at room temperature, followed by reduction in 10 mM DTT (Sigma‐Aldrich) and alkylation in 40 mM iodoacetamide (Sigma‐Aldrich). The resin was washed, followed by on‐column protease digestion, and analyzed by LC‐MS/MS as described below. To exclude the nonspecific binding proteins, competitive‐binding experiments were performed in which IR‐SCs were incubated with 5 μM PL for 2 hr then incubated with 5 μM PL probe for 3 hr. In parallel, IR‐SCs were initially incubated with 5 μM PL probe for 2 hr then incubated with 5 μM PL for 3 hr. We repeated the CTL and PL probe binding assays and proteomics analyses two to three times for each cell type. PL selective and specific targets were identified using the combination of an enrichment ratio of ≥2 (i.e., the average spectra reads in IR‐SCs and RE‐SCs were ≥2‐fold higher than those in NCs) and a PL competition ratio of ≥2 (i.e., the spectra reads on cells treated with the PL probe followed by PL were ≥2‐fold higher than those in the cells treated with PL followed by the PL probe), as described in the manuscript.

All raw files were analyzed together using software MaxQuant (Cox & Mann, 2008 ) (version 1.5.3.30) as described before (Keilhauer, Hein & Mann, 2015 ) with minor modification. The derived peak list was searched with the built‐in Andromeda search engine (Cox et al., 2011 ) against the reference human proteome downloaded from Uniprot ( http://www.uniprot.org/ ) on 04‐20‐2016 (176,494 sequences). Strict trypsin specificity was required with cleavage C‐terminal after K or R, allowing up to two missed cleavages. The minimum required peptide length was set to seven amino acids. Carbamidomethylation of cysteine was set as a fixed modification, and N‐acetylation of proteins N termini and oxidation of methionine were set as variable modifications. As no labeling was performed, multiplicity was set to 1. During the main search, parent masses were allowed an initial mass deviation of 4.5 ppm, and fragment ions were allowed a mass deviation of 0.5 Da. PSM and protein identifications were filtered using a target‐decoy approach at a FDR of 1%. The second peptide feature was enabled. The match between runs option was also enabled with a match time window of 0.7 min and an alignment time window of 20 min. Relative, label‐free quantification of proteins was performed using the MaxLFQ algorithm (Cox et al., 2014 ) integrated into MaxQuant. The parameters were as follows: Minimum ratio count was set to 1, the FastLFQ option was enabled, LFQ minimum number of neighbors was set to 3, and the LFQ average number of neighbors was set to 6, as per default. The “ProteinGroups” output file from MaxQuant is available in the supplement (Supporting Information Table S1 ).

Western blot analysis was performed according to our previous publication (Chang et al., 2016 ). See the details in Supporting Information Data S1 .

Six hundred nanograms purified human recombinant OXR1 ( NM_181354 ) protein (Cat# TP760961, OriGene, Rockville, MD) was incubated with biotin‐labeled PL probe in the absence or presence of a fivefold excess of unlabeled PL. The mixture was separated by SDS‐PAGE, and an immunoblot was performed with antibodies against biotin (Streptavidin (HRP), cat# Ab7403, Abcam) to detect OXR1 bound to the biotin‐labeled PL probe. An immunoblot of OXR1 from each reaction was included as an input control.

Cells were lysed, and total RNA was extracted using an RNeasy Mini Kit (catalog no. 74106, Qiagen, Germantown, MD). RNA (1 μg) was reverse‐transcribed with the Superscript II first strand synthesis kit (Invitrogen, Germantown, MD). Real‐time PCR was performed using specific primers (Supporting Information Table S5 ) and the SYBR Green ROX Mix (Thermo Scientific). The sequence of the primers is given below. All experiments were performed in two to three replicates, and data were normalized to the housekeeping gene GAPDH .

NCs and IR‐SCs were incubated with 5 μM PL probe or vehicle for 5 hr, and Alexa Fluor azide 488 was added to detect the PL uptake efficiency following the Click‐It EdU imaging kit protocol ( C10337 , Thermo Fisher Scientific). The levels of PL in cells were visualized by microscopy then quantified by flow cytometry and ImageStream flow cytometry. For microscopy, images were taken with the ZEISS image system as described before (Chang et al., 2016 ). For flow cytometry, after fixing and staining, Alexa Fluor 488 conjugated to the PL probe inside the cells was measured with a BD LSR II flow cytometer (BD Biosciences, San Jose, CA). The MFI of Alexa Fluor 488‐conjugated PL probe was normalized by cell size which was measured with FSC measurement. For ImageStream flow cytometry, Alexa Fluor 488 conjugated to the PL probe was quantified with ImageStreamX Mark II System (Amnis, Merck Millipore, Germany) following the instructions from the manufacturer, and then normalized by cell size which was measured by the average area of cells.

ROS and apoptosis were measured with flow cytometry as previously described (Wang et al., 2016 ). See the details in Supporting Information Data S1 .

X(in)Z and SZ performed the majority of the experiments. GZ designed the PL and CTL probe. XL and X(uan)Z synthesized the probes. YW and JC assisted with some of the experiments. SGM and AJT performed the MS proteomics analysis. YH and DL performed gene ontology and pathway analyses. X(in)Z, SZ, RML, JC, JW, GZ, and DZ contributed to experimental design, data analysis, and manuscript preparation.