Development of an activity-based chemiluminogenic probe for γ-glutamylcyclotransferase.

While γ-glutamylcyclotransferase (GGCT) has been implicated in cancer-cell proliferation, the role of GGCT enzymatic activity in the regulation of cancer-cell growth remains unclear. Toward further understanding of GGCT in vivo, here we report a novel cell-permeable chemiluminogenic probe "MAM-LISA-103" that detects intracellular GGCT activity and apply it to in vivo imaging. We first developed a chemiluminogenic probe LISA-103, which simply and sensitively detects the enzymatic activity of recombinant GGCT through chemiluminescence. We then designed the cell-permeable GGCT probe MAM-LISA-103 and applied it to several biological experiments. MAM-LISA-103 successfully detected the intracellular GGCT activity in GGCT-overexpressing NIH-3T3 cells. Moreover, MAM-LISA-103 demonstrated tumor-imaging ability when administered to a xenograft model using immunocompromised mice inoculated with MCF7 cells.

Structural and Functional Analyses of Human ChaC2 in Glutathione Metabolism.

Glutathione (GSH) degradation plays an essential role in GSH homeostasis, which regulates cell survival, especially in cancer cells. Among human GSH degradation enzymes, the ChaC2 enzyme acts on GSH to form 5-l-oxoproline and Cys-Gly specifically in the cytosol. Here, we report the crystal structures of ChaC2 in two different conformations and compare the structural features with other known γ-glutamylcyclotransferase enzymes. The unique flexible loop of ChaC2 seems to function as a gate to achieve specificity for GSH binding and regulate the constant GSH degradation rate. Structural and biochemical analyses of ChaC2 revealed that Glu74 and Glu83 play crucial roles in directing the conformation of the enzyme and in modulating the enzyme activity. Based on a docking study of GSH to ChaC2 and binding assays, we propose a substrate-binding mode and catalytic mechanism. We also found that overexpression of ChaC2, but not mutants that inhibit activity of ChaC2, significantly promoted breast cancer cell proliferation, suggesting that the GSH degradation by ChaC2 affects the growth of breast cancer cells. Our structural and functional analyses of ChaC2 will contribute to the development of inhibitors for the ChaC family, which could effectively regulate the progression of GSH degradation-related cancers.

ChaC2, an Enzyme for Slow Turnover of Cytosolic Glutathione.

Glutathione degradation plays an important role in glutathione and redox homeostasis, and thus it is imperative to understand the enzymes and the mechanisms involved in glutathione degradation in detail. We describe here ChaC2, a member of the ChaC family of γ-glutamylcyclotransferases, as an enzyme that degrades glutathione in the cytosol of mammalian cells. ChaC2 is distinct from the previously described ChaC1, to which ChaC2 shows ∼50% sequence identity. Human and mouse ChaC2 proteins purified in vitro show 10-20-fold lower catalytic efficiency than ChaC1, although they showed comparable Km values (Km of 3.7 ± 0.4 mm and kcat of 15.9 ± 1.0 min-1 toward glutathione for human ChaC2; Km of 2.2 ± 0.4 mm and kcat of 225.2 ± 15 min-1 toward glutathione for human ChaC1). The ChaC1 and ChaC2 proteins also shared the same specificity for reduced glutathione, with no activity against either γ-glutamyl amino acids or oxidized glutathione. The ChaC2 proteins were found to be expressed constitutively in cells, unlike the tightly regulated ChaC1. Moreover, lower eukaryotes have a single member of the ChaC family that appears to be orthologous to ChaC2. In addition, we determined the crystal structure of yeast ChaC2 homologue, GCG1, at 1.34 Å resolution, which represents the first structure of the ChaC family of proteins. The catalytic site is defined by a fortuitous benzoic acid molecule bound to the crystal structure. The mechanism for binding and catalytic activity of this new enzyme of glutathione degradation, which is involved in continuous but basal turnover of cytosolic glutathione, is proposed.

RipAY, a Plant Pathogen Effector Protein, Exhibits Robust γ-Glutamyl Cyclotransferase Activity When Stimulated by Eukaryotic Thioredoxins.

The plant pathogenic bacterium Ralstonia solanacearum injects more than 70 effector proteins (virulence factors) into the host plant cells via the needle-like structure of a type III secretion system. The type III secretion system effector proteins manipulate host regulatory networks to suppress defense responses with diverse molecular activities. Uncovering the molecular function of these effectors is essential for a mechanistic understanding of R. solanacearum pathogenicity. However, few of the effectors from R. solanacearum have been functionally characterized, and their plant targets remain largely unknown. Here, we show that the ChaC domain-containing effector RipAY/RSp1022 from R. solanacearum exhibits γ-glutamyl cyclotransferase (GGCT) activity to degrade the major intracellular redox buffer, glutathione. Heterologous expression of RipAY, but not other ChaC family proteins conserved in various organisms, caused growth inhibition of yeast Saccharomyces cerevisiae, and the intracellular glutathione level was decreased to ∼30% of the normal level following expression of RipAY in yeast. Although active site mutants of GGCT activity were non-toxic, the addition of glutathione did not reverse the toxicity, suggesting that the toxicity might be a consequence of activity against other γ-glutamyl compounds. Intriguingly, RipAY protein purified from a bacterial expression system did not exhibit any GGCT activity, whereas it exhibited robust GGCT activity upon its interaction with eukaryotic thioredoxins, which are important for intracellular redox homeostasis during bacterial infection in plants. Our results suggest that RipAY has evolved to sense the host intracellular redox environment, which triggers its enzymatic activity to create a favorable environment for R. solanacearum infection.

Botch is a γ-glutamyl cyclotransferase that deglycinates and antagonizes Notch.

Botch promotes embryonic neurogenesis by inhibiting the initial S1 furin-like cleavage step of Notch maturation. The biochemical process by which Botch inhibits Notch maturation is not known. Here, we show that Botch has γ-glutamyl cyclotransferase (GGCT) activity that deglycinates Notch, which prevents the S1 furin-like cleavage. Moreover, Notch is monoglycinated on the γ-glutamyl carbon of glutamate 1,669. The deglycinase activity of Botch is required for inhibition of Notch signaling both in vitro and in vivo. When the γ-glutamyl-glycine at position 1,669 of Notch is degylcinated, it is replaced by 5-oxy-proline. These results reveal that Botch regulates Notch signaling through deglycination and identify a posttranslational modification of Notch that plays an important role in neurogenesis.

Probing the specificity of gamma-glutamylamine cyclotransferase: an enzyme involved in the metabolism of transglutaminase-catalyzed protein crosslinks.

γ-Glutamylamine cyclotransferase (gGACT) catalyzes the intramolecular cyclization of a variety of L-γ-glutamylamines producing 5-oxo-L-proline and free amines. Its substrate specificity implicates it in the downstream metabolism of transglutaminase products, and is distinct from that of γ-glutamyl cyclotransferase which acts on L-γ-glutamyl amino acids. To elucidate the mechanism by which gGACT distinguishes between L-γ-glutamylamine and amino acid substrates, the specificity of the rabbit kidney enzyme for the amide region of substrates was probed through the kinetic analysis of a series of L-γ-glutamylamines. The isodipeptide N(ε)-(L-γ-glutamyl)-L-lysine 1 was used as a reference. The kinetic constants of the L-γ-glutamyl derivative of n-butylamine 7, were nearly identical to those of 1. Introduction of a methyl or carboxylate group on the carbon adjacent to the side-chain amide nitrogen in L-γ-glutamylamine substrates resulted in a dramatic decrease in substrate properties for gGACT thus providing an explanation of why gGACT does not act on L-γ-glutamyl amino acids except for L-γ-glutamylglycine. Placement of substituents on carbons further removed from the side-chain amide nitrogen in L-γ-glutamylamines restored activity for gGACT, and L-γ-glutamylneohexylamine 19 had a higher specificity constant (k(cat) /K(m)) than 1. gGACT did not exhibit any stereospecificity in the amide region of L-γ-glutamylamine substrates. In addition, analogues (26-30) with heteroatom substitutions for the γ methylene position of the L-γ-glutamyl moiety were examined. Several thiocarbamoyl derivatives of L-cysteine (28-30) were excellent substrates for gGACT.

Identification and characterization of gamma-glutamylamine cyclotransferase, an enzyme responsible for gamma-glutamyl-epsilon-lysine catabolism.

Gamma-glutamylamine cyclotransferase (GGACT) is an enzyme that converts gamma-glutamylamines to free amines and 5-oxoproline. GGACT shows high activity toward gamma-glutamyl-epsilon-lysine, derived from the breakdown of fibrin and other proteins cross-linked by transglutaminases. The enzyme adopts the newly identified cyclotransferase fold, observed in gamma-glutamylcyclotransferase (GGCT), an enzyme with activity toward gamma-glutamyl-alpha-amino acids (Oakley, A. J., Yamada, T., Liu, D., Coggan, M., Clark, A. G., and Board, P. G. (2008) J. Biol. Chem. 283, 22031-22042). Despite the absence of significant sequence identity, several residues are conserved in the active sites of GGCT and GGACT, including a putative catalytic acid/base residue (GGACT Glu(82)). The structure of GGACT in complex with the reaction product 5-oxoproline provides evidence for a common catalytic mechanism in both enzymes. The proposed mechanism, combined with the three-dimensional structures, also explains the different substrate specificities of these enzymes. Despite significant sequence divergence, there are at least three subfamilies in prokaryotes and eukaryotes that have conserved the GGCT fold and GGCT enzymatic activity.

The identification and structural characterization of C7orf24 as gamma-glutamyl cyclotransferase. An essential enzyme in the gamma-glutamyl cycle.

The hypothetical protein C7orf24 has been implicated as a cancer marker with a potential role in cell proliferation. We have identified C7orf24 as gamma-glutamyl cyclotransferase (GGCT) that catalyzes the formation of 5-oxoproline (pyroglutamic acid) from gamma-glutamyl dipeptides and potentially plays a significant role in glutathione homeostasis. In the present study we have identified the first cDNA clones encoding a gamma-glutamyl cyclotransferase. The GGCT gene is located on chromosome 7p14-15 and consists of four exons that span 8 kb. The primary sequence is 188 amino acids in length and is unlike any protein of known function. We crystallized functional recombinant gamma-glutamyl cyclotransferase and determined its structure at 1.7 A resolution. The enzyme is a dimer of 20,994-Da subunits. The topology of GGCT is unrelated to other enzymes associated with cyclotransferase-like activity. The fold was originally classified as "BtrG-like," a small family that only includes structures of hypothetical proteins from Mus musculus, Escherichia coli, Pyrococcus horikoshii, and Arabidopsis thaliana. Since this is the first member of this family with a defined function, we propose to refer to this structure as the gamma-glutamyl cyclotransferase fold. We have identified a potential active site pocket that contains a highly conserved glutamic acid (Glu(98)) and propose that it acts as a general acid/base in the reaction mechanism. Mutation of Glu(98) to Ala or Gln completely inactivates the enzyme without altering the overall fold.