In Vivo Tagging and Characterization of S-Glutathionylated Proteins by a Chemoenzymatic Method**

Glutathione (GSH), a sulfhydryl-containing tripeptide present in most organisms at millimolar levels, plays a crucial role in redox homeostasis. Reactive cysteine residues are vulnerable to reactive oxygen or nitrogen species and thus depend heavily on GSH to avoid irreversible oxidation. Reversible conjugation of GSH to proteins through the formation of mixed disulfide bonds is termed protein glutathionylation (PSSG), which additionally alters or regulates protein functions in biological processes, including energy metabolism, signal transduction, ion transport, cytoskeletal assembly, and protein folding. Although various possible mechanisms have been proposed for PSSG, the delineation of its functional consequences in vivo remains a longstanding challenge owing to lack of appropriate tools to globally identify this important modification with high sensitivity. A conventional method to detect PSSG relies on the metabolic labeling of endogenous GSH with radioactive Scysteine and subsequent phosphor imaging on the gel obtained from 2D PAGE. However, this approach does not distinguish different types of S-thiolation, for example, cysteinylation from glutathionylation. Another challenge is the shortage of straightforward methods to enrich and identify these modified proteins. Although commercially available anti-GSH antibodies have been applied to detect PSSG, serious concern has been raised regarding their specificity and sensitivity. Recently, glutaredoxin-dependent biotin-switch methods were developed for efficient detection and enrichment of GSH-modified proteins. This method, however, requires alkylation of reduced cysteine residues, followed by reduction of GSH-modified cysteines with bacterial glutaredoxin and subsequent biotin labeling. Several concerns have been raised, such as incomplete reduction and alkylation, the specificity of bacterial glutaredoxin, and possible oxidations in cysteine residues of proteins owing to ambient exposure. Biotinylated GSH disulfide (biotin-GSSG) has also been used to directly label protein cysteine residues. The membrane permeability of biotin-GSSG enables intracellular formation of mixed sulfides. Although this method is suitable for detection and enrichment, the external addition of biotin-GSSG likely alters the normal thiol content and the GSH/GSSG ratio given that GSSG usually accounts for less than 1% of the total GSH content in mammalian cells. Furthermore, since the action of GSSG is not the major route of protein glutathionylation under physiological conditions, such a method may exclude an unexpectedly large pool of proteins during analysis. Herein we developed a selective and rapid method for detecting and identifying PSSG in mammalian cells by using biotinyl spermine (biotin-spm) and E. coli glutathionylspermidine synthetase (GspS; Figure 1a,b). The enzyme GspS catalyzes the amide bond formation between GSH and spermidine to generate glutathionylspermidine (Gsp; Figure 1 in the Supporting Information), a unique GSH derivative found only in a few Gram-negative bacteria and protozoa. We previously discovered that Gsp behaves similarly to GSH in forming disulfide bonds with cysteine residues of proteins in vivo. Consequently, if the enzyme GspS is expressed in mammalian cells and generates Gsp, the mixed-disulfide bond formation between Gsp and protein cysteine residues would be akin to in situ protein glutathionylation. The DNA fragment of the GspS, corresponding to the amino acid sequence of the Gsp synthetase domain, was incorporated to the pCMV2B vector to construct pCMV2BGspS. The introduction of the vector pCMV2B-GspS into human embryonic kidney (HEK) 293T cells allowed expression of the enzyme GspS and the subsequent conversion of endogenous GSH to Gsp in vivo (Figure 2). Based on the structure of the enzymeGspS in complex with substrate (PDB code: 2IOA), one NH2 group of spermidine is near the [*] Dr. B.-Y. Chiang, Dr. C.-C. Chou, F.-T. Hsieh, Dr. S. Gao, J. C.-Y. Lin, S.-H. Lin, T.-C. Chen, Prof. K.-H. Khoo, Prof. C.-H. Lin Institute of Biological Chemistry, Academia Sinica No. 128, Academia Road Section 2, Nan-Kang, Taipei, 11529 (Taiwan) and Institute of Biochemical Sciences National Taiwan University (Taiwan) E-mail: [email protected] [email protected] Dr. C.-C. Chou Core Facilities for Protein Structural Analysis Academia Sinica (Taiwan)