Substoichiometric hydroxynonenylation of a single protein recapitulates whole-cell-stimulated antioxidant response.
ABSTRACT: Lipid-derived electrophiles (LDEs) that can directly modify proteins have emerged as important small-molecule cues in cellular decision-making. However, because these diffusible LDEs can modify many targets [e.g., >700 cysteines are modified by the well-known LDE 4-hydroxynonenal (HNE)], establishing the functional consequences of LDE modification on individual targets remains devilishly difficult. Whether LDE modifications on a single protein are biologically sufficient to activate discrete redox signaling response downstream also remains untested. Herein, using T-REX (targetable reactive electrophiles and oxidants), an approach aimed at selectively flipping a single redox switch in cells at a precise time, we show that a modest level (∼34%) of HNEylation on a single target is sufficient to elicit the pharmaceutically important antioxidant response element (ARE) activation, and the resultant strength of ARE induction recapitulates that observed from whole-cell electrophilic perturbation. These data provide the first evidence that single-target LDE modifications are important individual events in mammalian physiology.
Project description:Redox imbalance in cells induces lipid peroxidation and generates a class of highly reactive metabolites known as lipid-derived electrophiles (LDEs) that can modify proteins and affects their functions. Identifying targets of LDEs is critical to understand how such modifications are functionally implicated in oxidative-stress associated diseases. Here we report a quantitative chemoproteomic method to globally profile protein targets and sites modified by LDEs. In this strategy, we designed and synthesized an alkyne-functionalized aminooxy probe to react with LDE-modified proteins for imaging and proteomic profiling. Using this probe, we successfully quantified >4000 proteins modified by 4-hydroxy-2-nonenal (HNE) of high confidence in mammalian cell lysate and combined with a tandem-orthogonal proteolysis activity-based protein profiling (TOP-ABPP) strategy, we identified ~400 residue sites targeted by HNE including reactive cysteines in peroxiredoxins, an important family of enzymes with anti-oxidant roles. Our method expands the toolbox to quantitatively profile protein targets of endogenous electrophiles and the enlarged inventory of LDE-modified proteins and sites will contribute to functional elucidation of cellular pathways affected by oxidative stress.
Project description:Lipid-derived electrophiles (LDEs) are reactive metabolites, which can covalently modify proteins and DNA and regulate diverse cellular processes. 2- trans-Hexadecenal (2-HD) is a byproduct of sphingolipid metabolism, involved in cytoskeletal reorganization, DNA damage, and apoptosis. In addition, the loss of ALDH3A2, an enzyme removing 2-HD in cells, is responsible for Sjörgen-Larsson Syndrome (SJS), suggesting that accumulation of 2-HD could lead to pathogenesis. However, the targets and the precise mechanisms of 2-HD are not well characterized. Herein, we report an alkyne-2-HD derivative as a bioorthogonal probe to explore the functions of 2-HD. We identified more than 500 potential cellular targets. Among them, the pro-apoptotic protein Bax can be covalently modified by 2-HD directly at the conserved Cys62 residue. Our work provided new chemical tools to explore the cellular functions of LDEs and revealed new mechanistic insights of the deregulation of lipid metabolism in diseases.
Project description:Through a "tag-and-modify" protein chemical modification strategy, we site-selectively phosphorylated the activation loop of protein kinase p38?. Phosphorylation at natural (180) and unnatural (172) sites created two pure phospho-forms. p38? bearing only a single phosphocysteine (pCys) as a mimic of pThr at 180 was sufficient to switch the kinase to an active state, capable of processing natural protein substrate ATF2; 172 site phosphorylation did not. In this way, we chemically recapitulated triggering of a relevant segment of the MAPK-signaling pathway in vitro. This allowed detailed kinetic analysis of global and stoichiometric phosphorylation events catalyzed by p38? and revealed that site 180 is a sufficient activator alone and engenders dominant mono-phosphorylation activity. Moreover, a survey of kinase inhibition using inhibitors with different (Type I/II) modes (including therapeutically relevant) revealed unambiguously that Type II inhibitors inhibit phosphorylated p38? and allowed discovery of a predictive kinetic analysis based on cooperativity to distinguish Type I vs II.
Project description:Prototypical electrophiles such as the lipid 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) are well recognized for their therapeutic potential. Electrophiles modify signalling proteins in both the cytosol and mitochondrion, which results in diverse cellular responses, including cytoprotective effects and, at high doses, cell death. These findings led us to the hypothesis that targeting electrophiles to specific compartments in the cell could fine-tune their biological effects. To examine this, we synthesized a novel mitochondrially targeted analogue of 15d-PGJ2 (mito-15d-PGJ2) and tested its effects on redox cell signalling. Mito-15d-PGJ2 caused profound defects in mitochondrial bioenergetics and mitochondrial membrane depolarization when compared with 15d-PGJ2. We also found that mito-15d-PGJ2 modified different members of the electrophile-responsive proteome, was more potent at initiating intrinsic apoptotic cell death and was less effective than 15d-PGJ2 at up-regulating the expression of HO-1 (haem oxygenase-1) and glutathione. These results demonstrate the feasibility of modulating the biological effects of electrophiles by targeting the pharmacophore to mitochondria.
Project description:Intertidal limpets are important grazers along rocky coastlines worldwide that not only control algae but also influence invertebrates such as common barnacles. For instance, grazing limpets ingest settling barnacle cyprid larvae (hereafter cyprids) and push cyprids and barnacle recruits off the substrate. Such limpet disturbance effects (LDEs) can limit barnacle recruitment, a key demographic variable affecting barnacle population establishment and persistence. In this study, we examined limpet (Lottia cassis) disturbance to barnacle (Chthamalus dalli, Balanus glandula) recruitment on the Pacific coast of Hokkaido, Japan, as information on limpet-barnacle interactions from this region is missing. We investigated, for the first time, whether barnacle size and recruitment intensity influence LDEs on barnacle recruitment. Small barnacles may be less susceptible to LDEs than larger barnacles, because small size may reduce the propbability of limpet disturbance. Moreover, recruitment intensity can influence LDEs, as high recruitment can compensate for LDEs on barnacle recruitment density. In Hokkaido, C. dalli cyprids are smaller than B. glandula cyprids, and C. dalli recruitment is higher than B. glandula recruitment. Thus, we hypothesized that LDEs on C. dalli recruitment would be weaker than those on B. glandula recruitment. To test our hypothesis, we conducted a field experiment during which we manipulated limpet presence/absence on the interior surfaces of ring-shaped cages. After four weeks, we measured barnacle recruitment and recruit size on the interior surfaces of the cages and found negative LDEs on C. dalli and B. glandula recruitment and recruit size. As hypothesized, the LDEs on C. dalli recruitment were weaker than the LDEs on B. glandula recruitment. Additionally, C. dalli recruits were smaller than B. glandula recruits. However, the LDEs on C. dalli recruit size were as strong as the LDEs on B. glandula recruit size, indicating that the smaller C. dalli recruits are not less susceptible to LDEs than B. glandula recruits. Since C. dalli recruitment was higher than B. glandula recruitment, we propose that the higher C. dalli recruitment compensated for the LDEs on C. dalli recruitment. Our findings indicate that the detected differences in LDEs on barnacle recruitment are related to barnacle recruitment intensity but not recruit size.
Project description:Despite the known propensity of small-molecule electrophiles to react with numerous cysteine-active proteins, biological actions of individual signal inducers have emerged to be chemotype-specific. To pinpoint and quantify the impacts of modifying one target out of the whole proteome, we develop a target-protein-personalized "electrophile toolbox" with which specific intracellular targets can be selectively modified at a precise time by specific reactive signals. This general methodology, T-REX (targetable reactive electrophiles and oxidants), is established by (1) constructing a platform that can deliver a range of electronic and sterically different bioactive lipid-derived signaling electrophiles to specific proteins in cells; (2) probing the kinetics of targeted delivery concept, which revealed that targeting efficiency in cells is largely driven by initial on-rate of alkylation; and (3) evaluating the consequences of protein-target- and small-molecule-signal-specific modifications on the strength of downstream signaling. These data show that T-REX allows quantitative interrogations into the extent to which the Nrf2 transcription factor-dependent antioxidant response element (ARE) signaling is activated by selective electrophilic modifications on Keap1 protein, one of several redox-sensitive regulators of the Nrf2-ARE axis. The results document Keap1 as a promiscuous electrophile-responsive sensor able to respond with similar efficiencies to discrete electrophilic signals, promoting comparable strength of Nrf2-ARE induction. T-REX is also able to elicit cell activation in cases in which whole-cell electrophile flooding fails to stimulate ARE induction prior to causing cytotoxicity. The platform presents a previously unavailable opportunity to elucidate the functional consequences of small-molecule-signal- and protein-target-specific electrophilic modifications in an otherwise unaffected cellular background.
Project description: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/non-enzyme-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 "G-REX"-a technique that allows controlled release of reactive electrophiles in vivo. Mitigating toxicity/off-target effects associated with uncontrolled bolus exposure, G-REX tagged first-responding innate cysteines that bind electrophiles under true kcat/Km conditions. G-REX identified two allosteric ubiquitin-conjugating proteins-Ube2V1/Ube2V2-sharing a novel privileged-sensor-cysteine. This non-enzyme-catalyzed-PTM triggered responses specific 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.
Project description:A redox-neutral palladium(II)-catalyzed conversion of aryl, heteroaryl, and alkenyl boronic acids into sulfinate intermediates, and onwards to sulfones and sulfonamides, has been realized. A simple Pd(OAc)2 catalyst, in combination with the sulfur dioxide surrogate 1,4-diazabicyclo[2.2.2]octane bis(sulfur dioxide) (DABSO), is sufficient to achieve rapid and high-yielding conversion of the boronic acids into the corresponding sulfinates. Addition of C- or N-based electrophiles then allows conversion into sulfones and sulfonamides, respectively, in a one-pot, two-step process.
Project description:We present a redox-neutral method for the photocatalytic generation of carbanions. Benzylic carboxylates are photooxidized by single electron transfer; immediate CO2 extrusion and reduction of the in situ formed radical yields a carbanion capable of reacting with aliphatic aldehydes as electrophiles giving the Grignard analogous reaction product.
Project description:Cholesterol-core nanoparticles (LDE) have been shown to be recognized by low-density lipoprotein receptors (LDLR) after administration; therefore, LDE is an ideal vehicle to deliver drug with targeting property. Paclitaxel, when incorporated into LDE, promotes atherosclerosis regression with reduced drug toxicity in rabbits through LDLR. Here, we tested whether LDE-paclitaxel could still be effective in reducing diet-induced atherosclerosis in a mouse model without LDLR. Nineteen LDLR knockout male mice were fed 1% cholesterol for 12 weeks. Then, 12 animals received 4-weekly intraperitoneal LDE-paclitaxel (4 mg/kg) while 7 controls received saline solution. On week 12 and 16, in vivo MRI of the aortic roots was performed. Aorta macroscopy was made after euthanasia. Reduction of atherosclerotic lesions was observed. LDE-paclitaxel treatment resulted in reduction of wall area (14%) and stenosis (22%) by MRI and 33% by macroscopy. Thus, LDE-paclitaxel may produce pharmacological effects through LDE uptake by mechanisms other than LDLR.