Photoinducible Oncometabolite Detection

Kulkarni, Rhushikesh A.; Briney, Chloe A.; Crooks, Daniel R.; Bergholtz, Sarah E.; Mushti, Chandrasekhar; Lockett, Stephen; Lane, Andrew N.; Fan, Teresa W.‐M.; Swenson, Rolf E.; Linehan, W. Marston; Meier, Jordan L.

doi:10.1002/cbic.201800651

Cited by 15 publications

(8 citation statements)

References 46 publications

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“…Comparative metabolomic analyses of these different models, and the development of improved tools to assess subcellular metabolite levels should aid in the future definition of this phenomenon. [37][38] An ancillary insight from our analysis is the distinct information available from studying the consequences of FH rescue (as was done in this study) versus studying the conditions of FHdependent transformation. For example, our whole proteome analyses unexpectedly found glycolytic enzymes become even more highly expressed in UOK268 cells upon FH rescue, suggesting that, at least in this model, the Warburg effect may not be completely reversible by re-introduction of FH alone.…”

Section: Discussionmentioning

confidence: 97%

Heterogeneous adaptation of cysteine reactivity to a covalent oncometabolite

Perez

Bak

Bergholtz

et al. 2020

Journal of Biological Chemistry

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An important context in which metabolism influences tumorigenesis is the genetic cancer syndrome hereditary leiomyomatosis and renal cell carcinoma (HLRCC), a disease in which mutation of the TCA cycle enzyme fumarate hydratase (FH) causes hyperaccumulation of fumarate. This electrophilic oncometabolite can alter gene activity at the level of transcription, via reversible inhibition of epigenetic dioxygenases, as well as posttranslationally, via covalent modification of cysteine residues. To better understand the potential for metabolites to influence posttranslational modifications important to tumorigenesis and cancer cell growth, here we report a chemoproteomic analysis of a kidney-derived HLRCC cell line. Using a general reactivity probe, we generated a dataset of proteomic cysteine residues sensitive to the reduction in fumarate levels caused by genetic re-introduction of active FH into HLRCC cell lines. This revealed a broad upregulation of cysteine reactivity upon FH rescue, which evidence suggests is caused by an approximately equal proportion of transcriptional and posttranslational modification-mediated regulation. Gene ontology analysis highlighted several new targets and pathways potentially modulated by FH mutation. Comparison of the new dataset to prior studies highlights considerable heterogeneity in the adaptive response of cysteine-containing proteins in different models of HLRCC. This is consistent with emerging studies indicating the existence of cell and tissue-specific cysteine-omes, further emphasizing the need for characterization of diverse models. Our analysis provides a resource for understanding the proteomic adaptation to fumarate accumulation, and a foundation for future efforts to exploit this knowledge for cancer therapy.

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Section: Discussionmentioning

confidence: 97%

Heterogeneous adaptation of cysteine reactivity to a covalent oncometabolite

Perez

Bak

Bergholtz

et al. 2020

Journal of Biological Chemistry

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“…Because fumarate is an excellent alkene substrate and the cycloadduct is fluorescent, Meier and co-workers designed a tetrazole probe 106 to detect the oncometabolite fumarate (Scheme 25a). 47 Owing to the efficient photochemical generation of nitriles imines, this approach provides improved sensitivity compared to the previously reported hydrazonyl chloride probes. 98 Similarly, An et al developed a BODIPYlinked bithiophene-tetrazole 109 as an off-on fluorescence probe for the detection of hydrogen peroxide inside HeLa cells.…”

Section: Chemosensorsmentioning

confidence: 99%

“…Recently, Carell and co-workers exploited the hydrazonyl chlorides for bioorthogonal protein modification in aqueous media . The drawback of using hydrazonyl chloride as the nitrile imine precursor is that the hydrazonyl chloride is prone to hydrolysis in aqueous media . In contrast, tetrazoles exhibit excellent stability under aqueous conditions and provide rapid on-demand access to nitrile imines via photoirradiation.…”

Section: Tetrazole Photoclick Chemistrymentioning

confidence: 99%

Light-Triggered Click Chemistry

2020

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The merging of click chemistry with discrete photochemical processes has led to the creation of a new class of click reactions, collectively known as photoclick chemistry. These light-triggered click reactions allow the synthesis of diverse organic structures in a rapid and precise manner under mild conditions. Because light offers unparalleled spatiotemporal control over the generation of the reactive intermediates, photoclick chemistry has become an indispensable tool for a wide range of spatially addressable applications including surface functionalization, polymer conjugation and cross-linking, and biomolecular labeling in the native cellular environment. Over the past decade, a growing number of photoclick reactions have been developed, especially those based on the 1,3-dipolar cycloadditions and Diels–Alder reactions owing to their excellent reaction kinetics, selectivity, and biocompatibility. This review summarizes the recent advances in the development of photoclick reactions and their applications in chemical biology and materials science. A particular emphasis is placed on the historical contexts and mechanistic insights into each of the selected reactions. The in-depth discussion presented here should stimulate further development of the field, including the design of new photoactivation modalities, the continuous expansion of λ-orthogonal tandem photoclick chemistry, and the innovative use of these unique tools in bioconjugation and nanomaterial synthesis.

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“…Upon photoirradiation, the diaryl tetrazole can release N 2 and generate a nitrilimine dipole in situ, which undergoes a facile cyclization reaction with an alkene dipolarophile to afford a pyrazoline cycloadduct. The nitrilimine–alkene cycloaddition is an ultra rapid reaction and has been used in photoinduced protein labeling. − Moreover, Huisgen et al also reported that the nitrilimine dipole can react with thiophenol through nucleophilic addition . Recently, several studies reported that the nitrilimine intermediate generated from UV irradiation of tetrazole can react with nearby nucleophilic groups. − Inspired by these findings, we decided to develop a Ub probe containing a C-terminal tetrazole as a photocaged warhead.…”

Section: Introductionmentioning

confidence: 99%

Photocaged Cell-Permeable Ubiquitin Probe for Temporal Profiling of Deubiquitinating Enzymes

2020

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Photocaged cell-permeable ubiquitin probe holds promise in profiling the activity of cellular deubiquitinating enzymes (DUBs) with the much needed temporal control. Here we report a new photocaged cell-permeable ubiquitin probe that undergoes photoactivation upon 365 nm UV treatment and enables intracellular deubiquitinating enzyme profiling. We used a semisynthetic approach to generate modular ubiquitin-based probe containing a tetrazole-derived warhead at the C-terminus of ubiquitin and employed a cyclic polyarginine cell-penetrating peptide (cR10) conjugated to the N-terminus of ubiquitin via a disulfide linkage to deliver the probe into live cells. Upon 365 nm UV irradiation, the tetrazole group is converted to a nitrilimine intermediate in situ, which reacts with nearby nucleophilic cysteine residue from the DUB active site. The new photocaged cell-permeable probe showed good reactivity toward purified DUBs, including USP2, UCHL1, and UCHL3, upon photoirradiation. The Ub-tetrazole probe was also assessed in HeLa cell lysate and showed robust labeling only upon photoactivation. We further carried out protein profiling in intact HeLa cells using the new photocaged cell-permeable ubiquitin probe and identified DUBs captured by the probe using label-free quantitative (LFQ) mass spectrometry. Importantly, the photocaged cell-permeable ubiquitin probe captured DUBs specifically in respective G1/S and G2/M phases in synchronized HeLa cells. Moreover, using this probe DUBs were profiled at different time points following the release of HeLa cells from G1/S phase. Our results showed that photocaged cell-permeable probe represents a valuable new tool for achieving a better understanding of the cellular functions of DUBs.

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Photoinducible Oncometabolite Detection

Cited by 15 publications

References 46 publications

Heterogeneous adaptation of cysteine reactivity to a covalent oncometabolite

Heterogeneous adaptation of cysteine reactivity to a covalent oncometabolite

Light-Triggered Click Chemistry

Photocaged Cell-Permeable Ubiquitin Probe for Temporal Profiling of Deubiquitinating Enzymes

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