A dual-function chemical probe for detecting erasers of lysine lipoylation

doi:10.1007/s11705-021-2051-0

Front. Chem. Sci. Eng.

2022, Vol. 16

Issue (1) : 121-127 https://doi.org/10.1007/s11705-021-2051-0

COMMUNICATION

A dual-function chemical probe for detecting erasers of lysine lipoylation

Yusheng Xie^1,², Jie Zhang^1,², Liu Yang^1,², Qingxin Chen^1,², Quan Hao³, Liang Zhang^2,⁴, Hongyan Sun^1,²(

)

¹. Department of Chemistry, City University of Hong Kong, Hong Kong, China
². Key Laboratory of Biochip Technology, Biotech and Health Centre, City University of Hong Kong Shenzhen Research Institute, Shenzhen 518057, China
³. School of Biomedical Sciences, University of Hong Kong, Hong Kong, China
⁴. Department of Biomedical Science, College of Veterinary Medicine and Life Sciences, City University of Hong Kong, Hong Kong, China

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Abstract

Lysine lipoylation plays vital roles in cell metabolism and redox processes. For example, removal of lipoylation will decrease pyruvate dehydrogenase activity and affect the citric acid cycle. Despite the important functions of lysine lipoylation, the mechanisms for the addition and removal of this modification remain largely unexplored. Very few useful chemical tools are available to study the interactions of lysine lipoylation with its regulatory delipoylation proteins. For example, immunoaffinity purification-mass spectrometry is one of such tools, which highly relies on antibody efficiency and purification techniques. Single-step activity based fluorogenic probes developed by our groups and others is also an efficient method to study the deacylation activity. Affinity-based labeling probe using photo-cross-linker is a powerful platform to study the transient and dynamic interactions of peptide ligands with the interacting proteins. Herein, we have designed and synthesized a dual-function probe KTLlip for studying enzymatic delipoylation (eraser) activity and interaction of lysine lipoylation with the eraser at the same time. We show that KTLlip can be used as a useful tool to detect delipoylation as demonstrated by its ability to fluorescently label the eraser activity of recombinant Sirt2. We envision that the probe will help delineate the roles of delipoylation enzyme in biology.

Keywords dual-function fluorescent probe labeling photo-cross-linker lipoylation modification eraser sirtuin

Corresponding Author(s): Hongyan Sun

Just Accepted Date: 18 March 2021 Online First Date: 28 April 2021 Issue Date: 27 December 2021

Cite this article:

Yusheng Xie,Jie Zhang,Liu Yang, et al. A dual-function chemical probe for detecting erasers of lysine lipoylation[J]. Front. Chem. Sci. Eng., 2022, 16(1): 121-127.

URL:

https://academic.hep.com.cn/fcse/EN/10.1007/s11705-021-2051-0
https://academic.hep.com.cn/fcse/EN/Y2022/V16/I1/121

Fig.1 Scheme 1?? (a) Mechanism of our strategy to detect enzymatic delipoylation activity and label the eraser by dual-functional probe KTLlip; (b) Synthetic route of probe KTLlip (DIEA: N,N-Diisopropylethylamine; DCM: dichloromethane; EDCI: N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride; NHS: N-hydroxysuccinimide; DMF: N,N-dimethylformamide; ACN: acetonitrile; TFA: trifluoroacetic acid; HOBT: 1-hydroxybenzotriazole hydrate; NBD-F: 4-fluor-7-nitro-benzofurazan).

Fig.2 (a) Fluorescence spectra of KTLlip (10 μmol/L) with Sirt2 (40 ng/µL) under various conditions (l_ex = 480 nm); (b) fluorescence detection of KTLlip with different sirtuins.

Fig.3 Time-dependent study of KTLlip (10 μmol/L) incubated with Sirt2 (40 ng/µL) in HEPES buffer (20 mmol/L, pH= 8.0) with 200 μmol/L NAD⁺ at 37 °C (λ_ex = 480 nm).

Fig.4 (a) Concentration-dependent labeling of KTLlip (0, 1, 2.5, 5, 10 and 20 µmol/L) with recombinant Sirt2 (0.3 μg/lane) under 15-min of UV irradiation; (b) UV irradiation-time-dependent (2, 5, 10, 15 min) labeling of KTLlip (10 μmol/L) with recombinant Sirt2 (0.3 μg/lane); (c) labeling of Sirt2-spiked (2 μg) HEK293 lysate (20 μg) by KTLlip (10 μmol/L); (d) molecular structure of affinity-based probe KPlip for Klip; (e) comparison of labeling of Sirt2 (0.3 μg/lane) using KTLlip and KPlip probe.

1	M D Spalding, S T Prigge. Lipoic acid metabolism in microbial pathogens. Microbiology and Molecular Biology Reviews: MMBR, 2010, 74(2): 200–228 https://doi.org/10.1128/MMBR.00008-10
2	R A Mathias, T M Greco, A Oberstein, H G Budayeva, R Chakrabarti, E A Rowland, Y Kang, T Shenk, I M Cristea. Sirtuin 4 is a lipoamidase regulating pyruvate dehydrogenase complex activity. Cell, 2014, 159(7): 1615–1625 https://doi.org/10.1016/j.cell.2014.11.046
3	R N Perham. Swinging arms and swinging domains in multifunctional enzymes: catalytic machines for multistep reactions. Annual Review of Biochemistry, 2000, 69(1): 961–1004 https://doi.org/10.1146/annurev.biochem.69.1.961
4	E A Rowland, C K Snowden, I M Cristea. Protein lipoylation: an evolutionarily conserved metabolic regulator of health and disease. Current Opinion in Chemical Biology, 2018, 42: 76–85 https://doi.org/10.1016/j.cbpa.2017.11.003
5	P Naiyanetr, J D Butler, L Meng, J Pfeiff, T P Kenny, K G Guggenheim, R Reiger, K Lam, M J Kurth, A A Ansari, et al.. Electrophile-modified lipoic derivatives of PDC-E2 elicits anti-mitochondrial antibody reactivity. Journal of Autoimmunity, 2011, 37(3): 209–216 https://doi.org/10.1016/j.jaut.2011.06.001
6	T J Thekkumkara, L Ho, I D Wexler, G Pons, L Te-Chung, M S Patel. Nucleotide sequence of a cDNA for the dihydrolipoamide acetyltransferase component of human pyruvate dehydrogenase complex. FEBS Letters, 1988, 240(1-2): 45–48 https://doi.org/10.1016/0014-5793(88)80337-5
7	L Packer, E H Witt, H J Tritschler. Alpha-lipoic acid as a biological antioxidant. Free Radical Biology & Medicine, 1995, 19(2): 227–250 https://doi.org/10.1016/0891-5849(95)00017-R
8	H Moini, L Packer, N E L Saris. Antioxidant and prooxidant activities of α-lipoic acid and dihydrolipoic acid. Toxicology and Applied Pharmacology, 2002, 182(1): 84–90 https://doi.org/10.1006/taap.2002.9437
9	M A Holbert, R Marmorstein. Structure and activity of enzymes that remove histone modifications. Current Opinion in Structural Biology, 2005, 15(6): 673–680 https://doi.org/10.1016/j.sbi.2005.10.006
10	R H Houtkooper, E Pirinen, J Auwerx. Sirtuins as regulators of metabolism and healthspan. Nature Reviews. Molecular Cell Biology, 2012, 13(4): 225–238 https://doi.org/10.1038/nrm3293
11	M Watroba, D Szukiewicz. The role of sirtuins in aging and age-related diseases. Advances in Medical Sciences, 2016, 61(1): 52–62 https://doi.org/10.1016/j.advms.2015.09.003
12	M M Müller, T W Muir. Histones: at the crossroads of peptide and protein chemistry. Chemical Reviews, 2015, 115(6): 2296–2349 https://doi.org/10.1021/cr5003529
13	T Komatsu, Y Urano. Evaluation of enzymatic activities in living systems with small-molecular fluorescent substrate probes. Analytical Sciences, 2015, 31(4): 257–265 https://doi.org/10.2116/analsci.31.257
14	A Chalkiadaki, L Guarente. The multifaceted functions of sirtuins in cancer. Nature Reviews. Cancer, 2015, 15(10): 608–624 https://doi.org/10.1038/nrc3985
15	P Bheda, H Jing, C Wolberger, H Lin. The substrate specificity of sirtuins. Annual Review of Biochemistry, 2016, 85(1): 405–429 https://doi.org/10.1146/annurev-biochem-060815-014537
16	J Du, Y Zhou, X Su, J J Yu, S Khan, H Jiang, J Kim, J Woo, J H Kim, B H Choi, et al.. Sirt5 is a NAD-dependent protein lysine demalonylase and desuccinylase. Science, 2011, 334(6057): 806–809 https://doi.org/10.1126/science.1207861
17	H Jiang, S Khan, Y Wang, G Charron, B He, C Sebastian, J Du, R Kim, E Ge, R Mostoslavsky, et al.. SIRT6 regulates TNF-α secretion through hydrolysis of long-chain fatty acyl lysine. Nature, 2013, 496(7443): 110–113 https://doi.org/10.1038/nature12038
18	Z Liu, T Yang, X Li, T Peng, H C Hang, X D Li. Integrative chemical biology approaches to examine ‘erasers’ for protein lysine fatty-acylation. Angewandte Chemie International Edition, 2015, 54(4): 1149–1152 https://doi.org/10.1002/anie.201408763
19	Y Xie, L Chen, R Wang, J Wang, J Li, W Xu, Y Li, S Q Yao, L Zhang, Q Hao, H Sun. Chemical probes reveal sirt2’s new function as a robust “eraser” of lysine lipoylation. Journal of the American Chemical Society, 2019, 141(46): 18428–18436 https://doi.org/10.1021/jacs.9b06913
20	R Baba, Y Hori, S Mizukami, K Kikuchi. Development of a fluorogenic probe with a transesterification switch for detection of histone deacetylase activity. Journal of the American Chemical Society, 2012, 134(35): 14310–14313 https://doi.org/10.1021/ja306045j
21	D R Rooker, D Buccella. Real-time detection of histone deacetylase activity with a small molecule fluorescent and spectrophotometric probe. Chemical Science (Cambridge), 2015, 6(11): 6456–6461 https://doi.org/10.1039/C5SC02704G
22	I N Gober, M L Waters. Supramolecular affinity labeling of histone peptides containing trimethyllysine and its application to histone deacetylase assays. Journal of the American Chemical Society, 2016, 138(30): 9452–9459 https://doi.org/10.1021/jacs.6b02836
23	Y Xie, J Ge, H Lei, B Peng, H Zhang, D Wang, S Pan, G Chen, L Chen, Y Wang, et al.. Fluorescent probes for single-step detection and proteomic profiling of histone deacetylases. Journal of the American Chemical Society, 2016, 138(48): 15596–15604 https://doi.org/10.1021/jacs.6b07334
24	Z Li, P Hao, L Li, C Y Tan, X Cheng, G Y Chen, S K Sze, H M Shen, S Q Yao. Design and synthesis of minimalist terminal alkyne-containing diazirine photo-crosslinkers and their incorporation into kinase inhibitors for cell- and tissue-based proteome profiling. Angewandte Chemie International Edition, 2013, 52(33): 8551–8556 https://doi.org/10.1002/anie.201300683
25	B F Cravatt, A T Wright, J W Kozarich. Activity-based protein profiling: from enzyme chemistry to proteomic chemistry. Annual Review of Biochemistry, 2008, 77(1): 383–414 https://doi.org/10.1146/annurev.biochem.75.101304.124125
26	D K Nomura, M M Dix, B F Cravatt. Activity-based protein profiling for biochemical pathway discovery in cancer. Nature Reviews. Cancer, 2010, 10(9): 630–638 https://doi.org/10.1038/nrc2901

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