Linkage-Specific Synthesis of Di-ubiquitin Probes Enabled by the Incorporation of Unnatural Amino Acid ThzK

Overview

Journal Chembiochem

Specialty Biochemistry

Date 2022 Mar 9

PMID 35263494

Authors

Han Zhou

Tomaya Carpenter

Xuan Fu

Bo Jin

Britton Ody

Mohammad Sazid Hassan

Savannah E Jacobs

Jenny Cheung

Eve M Nicholson

Mark Turlington

Bo Zhao

Sonja Lorenz

T Ashton Cropp

Jun Yin

Affiliations

Soon will be listed here.

Abstract

Di-ubiquitin (diUB) conjugates of defined linkages are useful tools for probing the functions of UB ligases, UB-binding proteins and deubiquitinating enzymes (DUBs) in coding, decoding and editing the signals carried by the UB chains. Here we developed an efficient method for linkage-specific synthesis of diUB probes based on the incorporation of the unnatural amino acid (UAA) N -L-thiaprolyl-L-Lys (L-ThzK) into UB for ligation with another UB at a defined Lys position. The diUB formed by the UAA-mediated ligation reaction has a G76C mutation on the side of donor UB for conjugation with E2 and E3 enzymes or undergoing dethiolation to generate a covalent trap for DUBs. The development of UAA mutagenesis for diUB synthesis provides an easy route for preparing linkage-specific UB-based probes to decipher the biological signals mediated by protein ubiquitination.

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References

Kumar K, Spasser L, Erlich L, Bavikar S, Brik A . Total chemical synthesis of di-ubiquitin chains. Angew Chem Int Ed Engl. 2010; 49(48):9126-31. DOI: 10.1002/anie.201003763. View

Virdee S, Ye Y, Nguyen D, Komander D, Chin J . Engineered diubiquitin synthesis reveals Lys29-isopeptide specificity of an OTU deubiquitinase. Nat Chem Biol. 2010; 6(10):750-7. DOI: 10.1038/nchembio.426. View

Zhao X, Heng J, Guardavaccaro D, Jiang R, Pagano M, Guillemot F . The HECT-domain ubiquitin ligase Huwe1 controls neural differentiation and proliferation by destabilizing the N-Myc oncoprotein. Nat Cell Biol. 2008; 10(6):643-53. PMC: 2680438. DOI: 10.1038/ncb1727. View

Fottner M, Brunner A, Bittl V, Horn-Ghetko D, Jussupow A, Kaila V . Site-specific ubiquitylation and SUMOylation using genetic-code expansion and sortase. Nat Chem Biol. 2019; 15(3):276-284. DOI: 10.1038/s41589-019-0227-4. View

Xu L, Huang J, Chen C, Qu Q, Shi J, Pan M . Chemical Synthesis of Natural Polyubiquitin Chains through Auxiliary-Mediated Ligation of an Expressed Ubiquitin Isomer. Org Lett. 2017; 20(2):329-332. DOI: 10.1021/acs.orglett.7b03515. View

Ye Y, Rape M . Building ubiquitin chains: E2 enzymes at work. Nat Rev Mol Cell Biol. 2009; 10(11):755-64. PMC: 3107738. DOI: 10.1038/nrm2780. View

Edelmann M, Iphofer A, Akutsu M, Altun M, Di Gleria K, Kramer H . Structural basis and specificity of human otubain 1-mediated deubiquitination. Biochem J. 2008; 418(2):379-90. DOI: 10.1042/BJ20081318. View

Lorenz S, Bhattacharyya M, Feiler C, Rape M, Kuriyan J . Crystal Structure of a Ube2S-Ubiquitin Conjugate. PLoS One. 2016; 11(2):e0147550. PMC: 4734694. DOI: 10.1371/journal.pone.0147550. View

Oh E, Akopian D, Rape M . Principles of Ubiquitin-Dependent Signaling. Annu Rev Cell Dev Biol. 2018; 34:137-162. DOI: 10.1146/annurev-cellbio-100617-062802. View

10.

Wenzel D, Stoll K, Klevit R . E2s: structurally economical and functionally replete. Biochem J. 2010; 433(1):31-42. PMC: 3118098. DOI: 10.1042/BJ20100985. View

11.

Tang S, Liang L, Si Y, Gao S, Wang J, Liang J . Practical Chemical Synthesis of Atypical Ubiquitin Chains by Using an Isopeptide-Linked Ub Isomer. Angew Chem Int Ed Engl. 2017; 56(43):13333-13337. DOI: 10.1002/anie.201708067. View

12.

Li G, Liang Q, Gong P, Tencer A, Zhuang Z . Activity-based diubiquitin probes for elucidating the linkage specificity of deubiquitinating enzymes. Chem Commun (Camb). 2013; 50(2):216-8. PMC: 3918478. DOI: 10.1039/c3cc47382a. View

13.

Liu J, Li Y, Deol K, Strieter E . Synthesis of Branched Triubiquitin Active-Site Directed Probes. Org Lett. 2019; 21(17):6790-6794. PMC: 7007859. DOI: 10.1021/acs.orglett.9b02406. View

14.

Weber A, Elliott P, Pinto-Fernandez A, Bonham S, Kessler B, Komander D . A Linear Diubiquitin-Based Probe for Efficient and Selective Detection of the Deubiquitinating Enzyme OTULIN. Cell Chem Biol. 2017; 24(10):1299-1313.e7. PMC: 5658516. DOI: 10.1016/j.chembiol.2017.08.006. View

15.

Nguyen D, Elliott T, Holt M, Muir T, Chin J . Genetically encoded 1,2-aminothiols facilitate rapid and site-specific protein labeling via a bio-orthogonal cyanobenzothiazole condensation. J Am Chem Soc. 2011; 133(30):11418-21. DOI: 10.1021/ja203111c. View

16.

El Oualid F, Merkx R, Ekkebus R, Hameed D, Smit J, de Jong A . Chemical synthesis of ubiquitin, ubiquitin-based probes, and diubiquitin. Angew Chem Int Ed Engl. 2010; 49(52):10149-53. PMC: 3021723. DOI: 10.1002/anie.201005995. View

17.

Leung I, Dekel A, Shifman J, Sidhu S . Saturation scanning of ubiquitin variants reveals a common hot spot for binding to USP2 and USP21. Proc Natl Acad Sci U S A. 2016; 113(31):8705-10. PMC: 4978272. DOI: 10.1073/pnas.1524648113. View

18.

Li X, Fekner T, Ottesen J, Chan M . A pyrrolysine analogue for site-specific protein ubiquitination. Angew Chem Int Ed Engl. 2009; 48(48):9184-7. DOI: 10.1002/anie.200904472. View

19.

Weikart N, Sommer S, Mootz H . Click synthesis of ubiquitin dimer analogs to interrogate linkage-specific UBA domain binding. Chem Commun (Camb). 2011; 48(2):296-8. DOI: 10.1039/c1cc15834a. View

20.

Castaneda C, Liu J, Kashyap T, Singh R, Fushman D, Cropp T . Controlled enzymatic synthesis of natural-linkage, defined-length polyubiquitin chains using lysines with removable protecting groups. Chem Commun (Camb). 2011; 47(7):2026-8. PMC: 3190230. DOI: 10.1039/c0cc04868b. View