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Ube2V2 Is a Rosetta Stone Bridging Redox and Ubiquitin Codes, Coordinating DNA Damage Responses Yi Zhao,† Marcus J. C. Long,† Yiran Wang,† Sheng Zhang,‡ and Yimon Aye*,†,§ †
Department of Chemistry & Chemical Biology and ‡Proteomics and Mass Spectrometry Facility, Institute of Biotechnology, Cornell University, Ithaca, New York 14850, United States § Department of Biochemistry, Weill Cornell Medicine, New York, New York 10065, United States S Supporting Information *
ABSTRACT: Posttranslational modifications (PTMs) are the lingua franca of cellular communication. Most PTMs are enzyme-orchestrated. However, the reemergence of electrophilic drugs has ushered mining of unconventional/nonenzyme-catalyzed electrophile-signaling pathways. Despite the latest impetus toward harnessing kinetically and functionally privileged cysteines for electrophilic drug design, identifying these sensors remains challenging. Herein, we designed “GREX”a technique that allows controlled release of reactive electrophiles in vivo. Mitigating toxicity/off-target effects associated with uncontrolled bolus exposure, G-REX tagged f irst-responding innate cysteines that bind electrophiles under true kcat/Km conditions. G-REX identified two allosteric ubiquitin-conjugating proteinsUbe2V1/Ube2V2sharing a novel privileged-sensor-cysteine. This non-enzyme-catalyzedPTM triggered responses specif ic to each protein. Thus, G-REX is an unbiased method to identify novel functional cysteines. Contrasting conventional active-site/off-active-site cysteine-modifications that regulate target activity, modification of Ube2V2 allosterically hyperactivated its enzymatically active binding-partner Ube2N, promoting K63-linked client ubiquitination and stimulating H2AX-dependent DNA damage response. This work establishes Ube2V2 as a Rosetta-stone bridging redox and ubiquitin codes to guard genome integrity.
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constants (high kcat/Km). Unlike phosphorylation, ubiquitination is dominated by reactive thiol chemistry: Ub-conjugation proceeds through multiple enzyme-bound Ub-thioester intermediates. These conjugating enzymes are ROS-sensitive.13 Deubiquitinating/deSUMOylating enzymes (DUBs/SENPs) are mostly thiol-active proteases; many DUBs and SENPs are indeed targets of ROS. Many of these ROS adducts involve direct modification of the active-site cysteine residue that is privileged due to its low pKa and low kinetic barrier to reaction with ROS. In most cases, free thiol ushers regain in enzymatic activity. The Ub-proteasome pathway is RES-sensitive, although this is more nuanced than ROS-sensing. Many natural electrophiles, including prostaglandins,14 4-hydroxynonenal (HNE),15 and dietary isothiocyanates16 affect Ub-modification events (semi)specifically. Several RES target active-site or other important cysteines on Ub-activating (E1), -conjugating (E2), and -ligating (E3, HECT-type) enzymes. Regardless of the site, most RES-modifications are irreversiblean attribute of RES signaling that may improve efficacy/confer different latency/ longevity relative to ROS signaling.12
hrough a phenomenal research effort we now understand much about complex post-translational regulation in cell signaling. Approximately 10% of the genome is involved in phosphorylation1 and ubiquitination:2,3 a complex series of “codes” specific to both healthy and disease states is overseen by this suite of enzymes. Gaining a clearer comprehension of these paradigmatic signaling pathways has impacted several aspects of human health, including prophylaxis, diagnosis, drug design, and personal medicine. Recent years have witnessed success of kinome-targeting pharmaceuticals,4 and an intense pursuit of drug discovery is now also aimed at proteins/ pathways involving ubiquitination.5,6 No approved drugs currently target ubiquitin (Ub) conjugation/deconjugation,7 but the proteasomea molecular machine intrinsically linked to the ubiquitin pathwaysis a bona fide drug target.6 Against the backdrop of these exquisite enzyme-regulated signaling subsystems, the cell has also harnessed reactive smallmolecule signaling mediators to fine-tune responses. In this paradigm, reactive oxygen or electrophilic species (ROS/RES) directly modify a specific signal-sensing protein, preempting decision-making.8−11 Because ROS and RES exist at low levels during signaling, sensor residues on redox-responsive proteins are likely “kinetically privileged”, i.e., inherently tuned to rapidly react with specific ROS/RES12 with rapid second-order rate © XXXX American Chemical Society
Received: November 18, 2017
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DOI: 10.1021/acscentsci.7b00556 ACS Cent. Sci. XXXX, XXX, XXX−XXX
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Figure 1. G-REX identifies endogenous privileged first responder (PFR)-cysteines, including two novel sensors, Ube2V1 and Ube2V2, through its capability to genome-wide target-ID endogenous PFRs under electrophile-limited conditions. (A) General setup for G-REX. Treatment of HEK293T cells ectopically expressing HaloTag results in specific binding of the inert photocaged RES-precursor [e.g., Ht-PreHNE(alkyne)]. (In dotted box is the ribbon model of Halo bound to Ht-PreHNE(alkyne), chemical structure of which is shown in Figure S1B). Any unbound probe is washed out. Upon low-energy light exposure of cells (see methods section), this Halo−Ht-PreHNE(alkyne) complex releases a stoichiometric amount of HNE(alkyne) (t1/2 < 1−2 min) (red dot) within the microenvironment of Halo, enabling substoichiometric covalent tagging of native PFRs to HNE. Indicated established pulldown-proteomics analysis permits HT-target-ID genome-wide. (B) (Left) The indicated recombinant protein (12 μM) was treated with HNE-alkyne (12 μM) at 37 °C and aliquots were removed as a function of indicated time and diluted into Click reaction mixture containing Cy5-azide (reporting on HNEylation) and subsequently analyzed by Sypro-Ruby (total protein). (Right) Plot of total unlabeled protein remaining as a function of time based on band-intensity quantitation. Also see Figure S3A. (C) An illustrative model of N-terminal HaloTagged Ube2V2 complexed to Ube2N with the photocaged-precursor, Ht-PreHNE, bound at Halo. (See Figure S1A for general T-REX setup.) Ribbon structure is adapted from PDB: 1J7D. The newly discovered HNE-responsive C69 (this work) within Ube2V2, and the catalytic cysteine (C87) within Ube2N, are indicated. (D) MS/MS spectrum of a triply charged ion at m/z 795.75653+ identifying an HNE alkyne modified peptide: B
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IYSLKVECGPKYPEAPPSVR in which Cys69 of the UBE2 V2 protein digested by ArgC is modified by HNE alkyne with Xcorr score at 2.20 by Proteome Discoverer database search. The MS/MS spectrum was manually inspected and confirmed. The relative low Xcorr score is apparently due to the labile nature of the HNE alkyne which readily loses the HNE alkyne molecule from Cys69 residue during the CID fragmentation. This observation was supported by the two dominant ions (m/z 745.353+ and m/z 1117.372+) in the MS/MS spectrum representing the native targeted peptide (after loss of HNE alkyne). The inset shows the expanded view of MS survey scan for the precursor ion of the HNE-alkyne-modified peptide (m/z 795.75653+) with observed mass accuracy at 1.04 ppm. Also see Tables S1−S2. (E) HEK293T cells were transfected with the indicated plasmids, treated with Ht-PreHNE, and either exposed to light, or not irradiated. Normalized lysates from these two sets of cells were treated with either TEV-protease or buffer alone, respectively. Lysates were then subject to Click coupling with Cy5-azide, and analyzed by in-gel fluorescence for Cy5 signal. “1, 2, and N” respectively designate Ube2V1, Ube2V2, and Ube2N. Refer to Figure S4A for Cy5 gel and blots in full-view. (F) Similar to E but cells were cotransfected with either empty vector (−) or a plasmid of the same vector expressing HA-Ube2N (+). Region of interest in Cy5 gel is marked by a red rectangle. Refer to Figure S4B for Cy5 gel and blots in full-view. (G) Similar to F. Refer to Figure S4C for Cy5 gel and blots in full-view. M designates molecular weight marker lane in all gels/blots in this and all figures elsewhere.
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With the resurgence of electrophilic pharmacophores17 and the search for novel drug-targeting mechanisms,18 privileged RES-sensing residues and the proteins/pathways they control have come to the fore of disease treatment.19 Our recent work indicates that (i) there are specific subsets of orthogonal ROSand RES-sensing cysteines,20 meaning specific RES-sensors could be ideal foundations for electrophilic drug discovery;21 and (ii) privileged electrophile sensors may neither need to be active-site nucleophiles nor present in proteins with any specific chemical function. Hence RES-sensor identification could offer a pipeline to regulate pathway flux and modulate undruggable proteins without the difficulties associated with targeting activesite residues.22 Identification of bona fide sensor cysteines is difficult.23 An extensive series of innovative work has been done to identify electrophile-sensor proteins in cells, and some work has studied model organisms. There are two main strategies. The first is bolus exposure of cells with reactive electrophiles, followed by affinity capture of modified proteins and MS.24 This strategy excitingly identifies a huge number of targets but is dominated by mass action/hypermodification and is often prone to artifacts caused by end-point toxicity and perturbation of innate redox balance. A second strategy uses competitive profiling of a specific set of reactive residues.25 This innovative strategy is very powerful and more sensitive than bolus-dosing. While much of our current understanding in redox biology has been derived using these pioneering methods, some key limitations remain to be addressed.21 For instance, both of the above methods administer an excess of electrophile from outside of cells/animals; thus nuances of low-stoichiometry ontarget RES-modifications that drive phenotypically dominant redox responses at a specific time are often lost. Indeed, RES permeation into the cell, interaction with cellular redox machinery (e.g., glutathione), and buildup of metabolites are complex, time-dependent processes, rendering bulk RESexposure from outside of cells a far-from-controlled environment. This effect is magnified in whole organisms where phenotypic outputs from bulk exposure are a function of complex pharmacokinetics as well as amalgamation of on-target and off-target responses elicited by uncontrolled RES-exposure. Furthermore, as the competitive profiling method measures loss of labeling by the proxy (e.g., iodoacetamide), conclusions from the indirect measure of RES-modification may be confounded by off-target and/or secondary modifications/functional coupling: selective labeling of minor isoforms/complexes in low abundance may also be missed.
G-REX: AN UNBIASED METHOD TO PROFILE PRIVILEGED INNATE ELECTROPHILE-SENSOR PROTEINS To gain a new ability to directly assay downstream ramifications of these nonenzymatic redox-modification events at singleprotein-target resolution and obtain mechanistic information about precision RES-signaling, we recently developed “T-REX” (targetable reactive electrophiles and oxidants)26 (Figure S1A− B). While T-REX has provided the previously inaccessible ability to directly read-out functional consequences of targetspecific RES-modifications in vivo (cells, worms, and fish) in the backdrop of an otherwise unperturbed proteome, it has the following limitations: (1) T-REX relies on ectopic overexpression of Halo-fusion proteins, in which HaloTag is fused to individual select targets; (2) one thus has to have some prior knowledge about which targets could potentially bear functionally responsive cysteines [i.e., privileged first responders (PFRs)]. To that end, potential PFRs for a medium-throughput T-REX screen need to be cherry-picked from previously published hits obtained from profiling methods built on bulkRES-introduction from outside the cell; and (3) T-REX is a one-target-at-a-time, low/medium-throughput gel-based screen. To strive for a functional high-throughput screen at the whole-genome scale to target-ID PFRs, we here devised a new platform, “G-REX”, that captures PFR-cysteines directly in vivo, at a specific user-defined time (Figure 1A). Unlike T-REX, GREX (1) requires no reliance on ectopic overexpression of the target protein of interest (i.e., HaloTag ectopically expressed alone in cells with no transgene fused to it serves as an anchor to a cell/fish/worm-permeable bioinert photocaged RESprecursor that can liberate controlled amount of RES such as HNE on demand); (2) G-REX is thus “casting a net” for innate PFRs at the whole-genome scale in a high-throughput manner, at the user-defined time/dose/duration/locale; (3) in that capacity, G-REX is the first method to deliver a native RES signal of choice in situ; and (4) it selectively captures targets specifically under RES-limited conditions (thus targets likely represent those that undergo low-stoichiometric RES-modifications, i.e., likely to be PFRs), as opposed to identification of targets labeled by RES following bulk introduction of reactive signals from outside of cells/animals, which often is nondiscriminating in all parameters (time, locale, target, and extent of RES-occupancy on individual targets). Specifically herein, G-REX identifies PFR-cysteines via instantaneous release of a minimal amount of specific endogenous electrophile [e.g., 4-hydroxynonenal (HNE)a known signaling messenger that is cytotoxic and mutagenic at high concentrations8−10] in a cell (Figure 1A and Figure S1B). C
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analyses on samples not treated with light or Ht-PreHNE, which could be used to eliminate false-positive hits.
We chose HNE as a well-established model for native bioactive signaling electrophiles housing cysteine-reactive Michaelacceptor motifs prevalent in many successful covalent drugs.21 Ultimately different electrophile chemotypes are anticipated to have differential fingerprint patterns of privileged sensors. Because the amount of electrophile is low and release is rapid (t1/2 < 1−2 min), only the most sensitive cysteines can react before HNE metabolism (true kcat/Km-type conditions). Under the conditions deployed, based on the calculated intracellular concentration of ectopic Halo and photo-uncaging efficiency of the Ht-PreHNE probe bound to Halo, ∼5 μM of HNE is released to the cell during G-REX (Supporting Information, Figure S1C). We compared time-dependent HNE-labeling of the proteome following either G-REX or bulk HNE (5 μM) exposure to live HEK293T cells ectopically expressing HaloTag construct. These experiments revealed that in situ HNE release by G-REX afforded a larger degree of proteome labeling in the initial times relative to exogenous HNE treatment (Figure S1D). Time required for HNE permeation into cells may partly explain these differences. The G-REX systemwhen directly coupled to our T-REX single-protein-redox targeting26 (Figure S1A)presents a previously inaccessible two-in-one capability, simultaneously enabling (1) proteome-wide profiling, and (2) target-specific functional validations of novel sensors and phenotypically dominant responses specifically triggered as a direct result of low-occupancy on-target RES-modifications under electrophilelimited conditions in situ (Figure 1A and Figure S1A−B). Using this G-REX−T-REX double-tap strategy, we identified a novel privileged cysteine of conserved importance present in two proteins that acts as a redox−Ub signaling shunt modulating two disparate signaling pathways. Gel-based analysis coupled with streptavidin blot verified successful labeling of proteins from G-REX (Figure 1A and Figure S1E−F). Many redox-sensing proteins, such as Keap1, are unusually cysteine (Cys) rich. These proteins likely use mass action to improve their odds of being HNEylated and trigger downstream signaling. In addition, for Keap1, many Cys’s are functional sensors.27 Our recent findings make a strong case that sensing ability is not necessarily correlated with the number of Cys’s: we have found some HNE-sensing proteins/enzymessuch as Akt320 and small heat shock protein26contain unique sensor Cys’s and rely mostly upon kinetic privilege to sense endogenous RES such as HNE. To increase the odds of finding highly reactive yet cysteine-poor sensors, we focused on the low-molecular-weight (LMW) protein pool (Figure S1G). A 15−25-kDa region of the resultant gel (Figure S1G) was thus cut and HNE-labeled proteins were identified by MS. The top hit from this pool was Ube2V2 (Mms228)a Ub-conjugating protein-variant with a poorly understood role in DNA damage. Human Ube2V2 bears only one cysteine (C69) (Figure S2). A homologous protein, Ube2V1 (Uev129) was the fourth highest confidence hit (Figure S1H and Table S1). Ube2V1 bears three cysteines, one of which (C94) is analogous to C69 in Ube2V2 (Figure S2).30 Although neither of these proteins is reportedly redox-sensitive, remarkably, five other hits in the top 10 [peptidyl-prolyl cis− trans isomerase,31 ADP-ribosylation factor-332 and -4,32 nucleoside diphosphate kinase,33 and cofilin-134] were known HNE-sensors. This evidence suggests that G-REX is a sensitive method to identify first-responding sensor proteins. It also gave us further impetus to validate Ube2V1 and Ube2V2 as novel low-MW PFRs, without undertaking standard parallel MS
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G-REX IDENTIFIES A NOVEL CONSERVED CYSTEINE PRESENT IN TWO PRIVILEGED SENSORS IN HUMANS, UBE2V1 AND UBE2V2 Because Ube2V2 that contains only one cysteine (C69) was the top hit and it was also found together with a homologous protein Ube2V1 containing a similar cysteine, we hypothesized that Ube2V2(C69)/[Ube2V1(C94)] (Figure S2A) are privileged sensors. Alignments of human Ube2V1 and Ube2V2 with other vertebrate counterparts showed that C69 and C94 are both conserved from humans to yeast (Figure S2B−D). Neither cysteine is implicated in electrophile sensing. The analogous cysteine in Saccharomyces cerevisiae Ube2V2 (Mms2) is also not required for activity.35 The longest isoform of human Ube2V1 has two other cysteines, one of which is not conserved beyond chimpanzees, whereas the other is conserved to frogs (Figure S2B). Interestingly, both Ube-2 V1 and -2V2 modulate ubiquitination activity of Ube2N (a low confidence hit in our G-REX data). Ube2N is an established Ub-conjugating E2 enzyme that assembles K63-linked ubiquitin, unanchored, or anchored on target proteins.28,29 As opposed to proteasomal targeting, K63-linked ubiquitination plays a role in cell signaling, including NF-κB signaling29,36 and the DNA damage response (DDR).37−39 Intriguingly, Ube2V2/Ube2V1 both lack a catalytic cysteine required for E2-activity, whereas Ube2N houses a catalytically essential nucleophilic Cys(C87) required for E2-catalytic function. Indeed, Ube2N(C87) has also proven susceptible to nitro-furan-derived electrophiles and acrylonitrile sulfonates.40 However, direct comparison of the efficiency of HNEylation of Ube2V2 and Ube2N in vitro (Figure 1B) unambiguously demonstrated that HNE sensing is unique to Ube2V2. By contrast, Ube2V2(C69S)which we predicted to be a mutant unable to undergo HNEylation but otherwise similar to wild-type (wt)-Ube2V2lost sensing (Figure S3A− B). Circular dichroism analyses showed that both Ube2V2 wt and C69S adopted a similar secondary structure (Figure S3C). The in vitro ubiquitination assays validated that these recombinantly purified wt and C69S Ube2V2 were functional (Figure S3D). HNEylation of Ube2V2 also did not significantly perturb/destabilize the protein’s fold (Figure S3E). Our findings from G-REXtogether with these in vitro analysesthus raise the tantalizing possibility that Ube2V1/2 serve as novel signaling shunts bridging the human redoxome and ubiquitome, enabling “signal exchange” between a native redoxlinked signal (HNE) and a canonical enzyme-catalyzed signal (Ub). The in-cell-relevance of target- and site(C69)-specific HNEsensing within Ube2V2 was verified using our single-protein redox-targeting method, T-REX, in live cells (Figure S1A−B),26 followed by LC-MS/MS on Ube2V2 enriched from extracts (Figure 1C−D and Table S2). These data underscore the capability of a G-REX−T-REX coupled platform to identify bona fide HNE-sensors at the proteome scale, and subsequently interrogate the consequences of on-target electrophilic modification under conditions that mimic endogenous signaling. From our recent unbiased screens, approximately 10% of proteins can react with HNE under T-REX.20,26 Because TREX is built on a quasi-intramolecular delivery,41 the efficiency of this targeting process is not affected by protein expression.20,26,41 Regardless, expression of Ube2N and Ube2V2/Ube2V1 proteins was similar in our experiments D
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Figure 2. T-REX-assisted Ube2V2(C69)-specific HNEylation elicits K63-linked-polyubiquitination that is lost in the C69S mutant. (A) HNEylation of Ube2V1 (specifically of C94), but not Ube2V2, selectively elicits upregulation in NF-κB-signaling (mean ± s.e.m, n ≥ 2 independent biological replicates). (B) HEK293T cells expressing wt-Halo-(FLAG)-Ube2V2 or the C69S mutant were treated with the DUB-inhibitor PR619 and highmolecular-weight (HMW) polyubiquitylated-Ube2V2 was assessed by western blot. (C) HEK293T cells expressing wt-Halo-(FLAG)-Ube2V2 (or the C69S mutant) and HA-Ubiquitin were exposed to the indicated conditions. Halo-(FLAG)-Ube2V2 from these cells was enriched by FLAGimmunoprecipitation (IP), and resulting samples were analyzed using the indicated antibodies. Left panel = “Input lanes”; Right panel = “IPsamples”. (D) Similar experiment to C, except nonenriched whole cell lysates were analyzed using the indicated antibodies, and an additional sample from the use of no-alkyne-variant of Ht-PreHNE but otherwise treated under identical conditions was also analyzed. (E) Similar experiment to C except global Ub pools were precipitated using HA IP (right panel) (there is no change in Ub since Ub is being IP-ed).
and Ube2N were 15 ± 6%, 5 ± 2%, and 50% reduction in Ube2N levels (#16, #17) relative to both wt-lines and lines expressing nontargeted shRNAs, and a third shRNA (#18) gave weaker knockdown (Figure S7B−C). Ube2N-knockdown lines did not show perturbation in poly-Ub-pools [Figure S7B, Anti-Ub (endogenous) blot]. Lines with higher knockdown-efficiencies (#16, #17) showed significantly reduced HMW-ubiquitination of Ube2V2 following T-REX-assisted HNEylation (Figure 4A, Figure S7D−E). Line #18 showed weak suppression of polyubiquitination, consistent with Ube2N-dose-dependent regulation of Ube2V2-polyubiquitination (Figure 4A−B). These datain conjunction with Ube2V2(C69S) hypomorphismestablish that Ube2V2(C69)-specific electrophilic modification stimulates Ube2N-enzymatic activity.
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UBE2V2-(C69)HNEYLATION-CONTROLLED UBE2N STIMULATION DRIVES DDR SIGNALING We further tracked the physiologic ramifications of this HNEylation event by mapping, at the proteome-scale, perturbations in Ube2V2-interactome in response to Ube2V2specific electrophilic modification using SILAC−T-REX. In other words, we were motivated to profile altered associations as a result of Ube2V2-specific HNEylation. Targets identified in this experimental setup consist of Ube2V2-HNEylationdependent associating partner(s) of Ube2V2/Ube2N or other protein(s) strongly associated with Ube2V2. SILACin contrast to standard pulldown-proteomicswas employed to I
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Figure 5. Ube2V2-Specific HNEylation regulates γ-H2AX levels in zebrafish. (A) Casper zebrafish embryos were injected with mRNA-encoding Halo-(FLAG)-Ube2V2 at the 1−4 cell stage. Once injection was complete, eggs were either exposed to Ht-PreHNE or DMSO. After 24-hincubation, fish were washed and exposed to light. After dechorionation and deyolking at 4 °C, embryos were lysed, biotin was attached using Click chemistry by biotin-azide, and lysates were ethanol precipitated. After resolubilization, biotinylated [i.e., HNE(alkyne)-modified] proteins were pulled down using streptavidin and analyzed by western blot. (See Figure S1A for workflow; Clicking with biotin-azide.) (B) Similar experiment to A, but at 3-h postlight-exposure, fish were fixed, permeabilized, and analyzed by whole-mount immunofluorescence using indicated antibodies. Scale bars, 100 μm. (C) Quantitation of images in B [mean ± s.e.m., n = 69 (T-REX), n = 74 (Light alone), n = 64 (Ht-PreHNE alone), n = 69 (DMSO)]. J
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unchanged (Figures 2D, 3A, and 4A). Both the above results are consistent with our sequence alignments that showed conservation of the sensor cysteine (C69) from humans to fish (Figure S2C,D). Furthermore, T-REX-treated fish selectively showed upregulation in γ-H2AX (Figure 5B−C). On the other hand, treatment of embryos with mitomycin-C led to a high level of γ-H2AX-expression relative to untreated fish, but these fish were severely deformed or died during treatment (Figure 5B− C; Figure S9B). Although latencies and other confounding factors make the comparison between mitomycin-C treatment and T-REX indirect, this result at least suggests that REStargeting of Ube2V2 could constitute a noninvasive method to prime DDR, without eliciting severe genotoxic stress.
(Figure 4F and Figure S8A−B). This DNA-synthesis stall did not occur in Ube2N-depleted cells (Figure S8C). Slowing of the replication fork has been shown to upregulate γ-H2AX formation at the point of replication.59,60 We thus proceeded to assay for changes in γ-H2AX following T-REXdirected Ube2V2(C69)-HNEylation. Indeed, an approximately 2-fold increase in cellular γ-H2AX was observed following Ube2V2(C69)-targeted-HNEylation (Figure 4G; Figure S8D− F). γ-H2AX upregulation was transient: increased γ-H2AX could be measured up to 18-h post light-induced HNE targeting, but γ-H2AX reduced to basal after 24 h (Figure S8E). This behavior is consistent with a signaling/preconditioning response. Mitomycin-C upregulated γ-H2AX-upregulation by 3−4-fold (Figure 4H; Figure S8A,C),61,62 validating the assay and showing that the effect of HNEylation is biologicallyas well as statisticallysignificant. γ-H2AXupregulation required both Ube2V2(C69) and Ube2N because (1) no significant γ-H2AX upregulation was measured in cells expressing Ube2V2(C69S) mutant at any point over the time course (18-h) post T-REX (Figures 4G and S8E); and (2) notably, wt-Ube2V2-specific HNEylation was amorphic for γH2AX-upregulation in Ube2N-depleted background (Figure 4H). However, both backgrounds hypomorphic for T-REXspecific γ-H2AX-upregulation [i.e., Ube2V2(C69S) mutantexpressing cells and cells deficient of Ube2N] still upregulated γ-H2AX upon mitomycin-C treatment (Figure 4G−H; Figure S8D−F).
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CONCLUSION
In sum, we have devised a regimen to identify functional electrophile-sensor proteins and surgically interrogate their specific responses in the backdrop of an otherwise largely unperturbed cell or animal. Previous studies to model electrophilic signaling subsystems have made use of global HNE flooding. Here we take a different track: we configure a “low-occupancy modification” setting, in which a brief “pulse” of HNE is delivered in situ, such that only f irst responders (regardless of expression level) are tagged. This approach offers a much-needed user-controlled protocol to tag, and (when coupled to T-REX) road-test functional cysteines. Importantly, G-REX uses direct affinity capture of modif ied cysteines, as opposed to current strategies that detect loss of labeling. Thus, G-REX minimizes false positives associated with off-target reactivity. The G-REX regimen could readily be applied to target-ID of a specific electrophile; to identify new RES-sensors for drug discovery; or extended to identification of off-target proteins hit by electrophilic drugs and linking these specific events to phenotypes, such as drug-induced hepatotoxicity. We validated G-REX by investigating the in vitro and in cellspecific HNEylation effects of our highest confidence novel hits. In vitro, Ube2V2 showed highly efficient conjugating efficiency to HNE and stimulated activity of the cognate E2, Ube2N. In cells, remarkably, mono-HNEylation of either privileged sensor was suf f icient to elicit hyper-stimulation of Ube2V1- or Ube2V2specific downstream signaling. The outcomes from the chemically and kinetically competent Ube2V2-point mutant that cannot sense HNE (in vitro or cells) strikingly support this functional sensing and signal propagation. These data support an HNE-shunt mechanism, whereby flux through the ubiquitin signaling node, Ube2N, is elevated by HNEylation of its allosteric binding-partner, Ube2V2. We propose this new signaling currency exchange to be a functional pathway that operates as a surveillance and maintenance mechanism for DDR64 in response to a transient rise in cellular electrophile flux. These data indicate that redox signalingHNEylation of one regulatory protein (at a site with no “expected” reactivity)can affect ubiquitin signaling via a third-party enzyme containing a catalytic cysteine (Ube2N). Given that defects in DDR65 are a common source of heritable disease, it is likely that exquisite regulation by small-molecule electrophiles on specific pathways could be mined for drug discovery and biomedical benefits.
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DDR PRECONDITIONING OCCURS IN ZEBRAFISH EMBRYOS The role of γ-H2AX in DNA-damage checkpoint remains enigmatic, although evidence exists that H2AX is required to initiate several DNA-damage checkpoints. In light of the reported increase in ubiquitination of other proteins during DNA damage (such as PCNA), we postulated that HNEylation, specifically of Ube2V2, and subsequent increase in ubiquitination of downstream targets of Ube2N, like PCNA, retards the replication fork and ultimately promotes DDR-like responses. Although preconditioning is a common mechanism whereby RES-signals elicit beneficial cytoprotective responses to defend against or adapt to cellular stress, triggering of DDR is not one of the pathways known to function through this mechanism. We were thus compelled to draw functional relevance of this intriguing signal exchange mechanism to a whole-vertebrate system. We chose zebrafish (Danio rerio).63 Our phylogenetic analysis indicated that zebrafish Ube2V2 possess a cysteine analogous to C69 in humans (Figure S2C,D). We first demonstrated that ectopic human Ube2V2 senses bioactive electrophiles such as HNE in zebrafish. We expressed human HaloTagged-Ube2V2 in zebrafish by injecting embryos at the one-cell stage with in vitro transcribed mRNA, incubated embryos with Ht-PreHNE (1 μM) for 1 day in the dark, then washed away excess probe, exposed the live specimens to light (5 min, 365 nm at 0.3 mW/cm2). A portion of these samples was subjected to biotin-mediated enrichment of in situ HNEylated proteins. The remaining portion was incubated for 3 h post light exposure. Subsequently the fish were fixed and γ-H2AX levels were measured using whole-mount immunofluorescence. Ube2V2 was significantly HNEylated following T-REX in zebrafish (Figure 5A). Targeted HNEylation led to detectable levels of HMW HNEylated-Ube2V2 that was polyubiquitinated (Figure S9A). As in cell culture, global ubiquitin pools were K
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acscentsci.7b00556. Supplementary methods and statistical analysis. Supplementary Figures S1−S9; Supplementary Tables S1−S6 (PDF) Table S1 (XLSX1 and Table S3 XLSX2)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Yimon Aye: 0000-0002-1256-4159 Author Contributions
Y.Z. performed all the cell-based and in vitro biochemical assays. M.J.C.L. performed zebrafish microinjection and M.J.C.L. and Y.Z. collaborated on imaging and biochemical assays involving zebrafish. Y.W. assisted Y.Z. in small-molecule synthesis. S.Z. assisted in LC-MS/MS data analysis. Y.A., Y.Z., and M.J.C.L. analyzed the data. Y.A. wrote the paper with assistance from M.J.C.L. and Y.Z. Notes
The authors declare the following competing financial interest(s): Provisional patent for the G-REX method in filing stages (by Cornell University).
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ACKNOWLEDGMENTS Professors Todd Evans (Weill Cornell Medicine) and Joseph Fetcho (Cornell University) for early guidance and zebrafish techniques transfer; Nikki Gilbert and Brian Miller from the Fetcho laboratory for assistance with fish husbandry and breeding; Dr. Xuyu Liu for assistance with cell-based protein stability assays; Alexandra Van Hall-Beauvais for providing recombinant purified His6-Halo protein; Professor Brian Crane’s laboratory for the use of CD spectrometer; zebrafish husbandry and microinjection/imaging facility [NIH R01 NS026593, J. Fetcho (Cornell University)], Cornell NMR facility [NSF MRI: CHE-1531632, Y.A.], and Cornell Imaging Center [NIH 1S10RR025502, R. M. Williams (Cornell University)] for instrumentation. Instrumentation, personnel, and supplies are fully or partly supported by NIH New Innovator (1DP2GM114850), Beckman Young Investigator, NSF CAREER (CHE-1351400), Office of Naval Research (ONR) Young Investigator (specific to work involving zebrafish), and Sloan Fellowship programs (FG-2016-6379) (to Y.A.).
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