Regulation by physiological mechanisms
As a relatively nonspecific cross-linking enzyme that is abundant in intracellular as well as extracellular environments, TG2 activity must be tightly regulated. The three most well understood physiological regulators of its enzymatic activity are Ca2+ ions, guanine nucleotides, and the redox potential.
Binding of Ca2+ ions is essential for TG2 catalytic activity. One molecule of TG2 can bind up to six Ca2+ ions with an apparent overall dissociation constant of 90 μM, as demonstrated by equilibrium dialysis.8 Although most of these binding sites have not been directly visualized, mutagenesis studies suggest that they are distributed across the protein.9 In contrast, GTP or GDP binds TG2 at a unique, structurally defined site with a dissociation constant of 1.6 μM.10, 11 GTP-bound, catalytically inactive TG2 requires high (>1 mM) Ca2+ ion concentrations for activation, although one high-affinity site (Kd = 0.1–15 μM) has been identified by isothermal titration calorimetry9, 12 and visualized in the GTP-bound crystal structure.6
Because TG2 activity requires relatively high concentrations of calcium and is inhibited by GTP/GDP, intracellular TG2 is catalytically silent under normal physiological conditions. However, these allosteric regulatory features do not explain why extracellular TG2 is also predominantly inactive in most tissues and organs.13 This feature is influenced by the redox sensitivity of the protein. Early studies showed that TG2 is susceptible to reversible inactivation by the formation of an intramolecular disulfide bond between cysteine residues other than the active site Cys277.14, 15 More recently, the switch between the (active) reduced state and the (inactive) oxidized state of TG2 has been characterized.16, 17 This redox switch centers on a triad of cysteine residues, the vicinal Cys370/Cys371 and Cys230, which have an unusually high redox potential of −190 mV.16 Through mutation analysis and alkylation studies, Cys230 was identified as the key redox sensor. Under oxidizing conditions, the inter-strand disulfide-bond Cys230-Cys370 forms first and facilitates formation of the more stable Cys370-Cys371 disulfide bond that inactivates TG2.17 Interestingly, the oxidation of TG2 is dependent on the presence of calcium ions, and oxidized TG2 appears to adopt an open conformation.7, 17
In summary, Ca2+, guanine nucleotides, and the redox potential collaborate to maintain mammalian TG2 in at least three distinct states, depending on local conditions (Fig. 2). The two most abundant states—the GTP/GDP bound form and the Ca2+ bound oxidized form—are inactive, whereas catalytic activity is only expressed in the transient Ca2+-bound, reduced form. Under homeostatic conditions, the cytosol has a high concentration of GTP, a low redox potential, and extremely low (<1 μM) levels of free Ca2+. Consequently, the intracellular pool of TG2 is in a reduced, catalytically inactive state. An influx of calcium ions can then activate the enzyme, as demonstrated in apoptotic states.18, 19 Similar arguments are also applicable to the mitochondrial and nuclear pools of TG2, because free Ca2+ concentration is comparably low in those compartments under ordinary conditions. In contrast, the extracellular matrix in most organs (e.g., intestine, liver) has a considerably lower concentration of GTP or GDP, a relative abundance of Ca2+ ions, and an oxidizing environment. Consequently, a vast fraction of the extracellular TG2 reservoir is also inactive.13
Figure 2. The three states of TG2: Reduced, guanine nucleotide bound TG2 is closed and inactive. Upon dissociation of GTP and binding of Ca2+, the enzyme opens to the active conformation, which can then be oxidized to the open, inactive, disulfide-bonded form, a process that is reversed by thioredoxin (adapted from Refs. 16 and 17).
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Both thermodynamic and kinetic factors are responsible for activating extracellular TG2. The high redox potential of the vicinal disulfide bond poises TG2 favorably for reduction relative to other disulfide bonded extracellular proteins, as the local environment becomes hypoxic. Indeed, this may be the mechanism for hypoxia induced, TG2 catalyzed serotonylation of fibronectin in rodent models of pulmonary hypertension.20 In addition, and perhaps more importantly, selective recognition of TG2 by the protein cofactor thioredoxin 1 leads to highly efficient activation of oxidized TG2.16 The precise mechanisms for thioredoxin secretion into the extracellular matrix remain to be established, although at least one pro-inflammatory signal has been identified. Specifically, cultured monocytic and epithelial cells that are responsive to interferon-γ secrete thioredoxin in response to elevated levels of this pro-inflammatory cytokine.16
Analogous to the recognition of oxidized TG2 by thioredoxin, other protein–protein interactions might also regulate TG2 activity in the extracellular matrix. Indeed, TG2 is known to interact with a number of extracellular proteins such as fibronectin, integrin, and calreticulin, as reviewed elsewhere.21 Although binding to fibronectin does not alter the enzymatic activity of TG2,22 one or more of the other protein partners could do so. Similarly, in the intracellular environment, GTP-bound TG2 has been shown to physically associate with other proteins. For example, in the presence of epinephrine, it associates with the α1-adrenergic receptor and induces phospholipase C activity.23 Given the inverse relationship between guanine nucleotide binding and transglutaminase activity, this G-protein like activity of TG2 may also be regulated by protein–protein interactions. Last but not least, noncovalent interactions between extracellular TG2 and cell surface carbohydrates can also modulate TG2 activity. For example, heparan sulfate has a binding site on the surface of the closed form of TG2.24, 25
In addition to the above allosteric mechanisms for regulating TG2, nature may have also evolved covalent modification strategies for regulating the catalytic activity of this mammalian enzyme. For example, protein kinase A catalyzes phosphorylation of Ser216 of human TG2, which in turn leads to activation of nuclear factor-κB, although the mechanistic basis of this phenomenon remains to be elucidated.26 Recent evidence also suggests that a small amount of a constitutively active proteolytic derivative of TG2 may be present in the cytosol, although the biochemical origins and physiological role of this fragment is unknown.27
Regulation by synthetic allosteric factors
To date, there have been several reports of small molecules that inhibit TG2 activity by binding to its active site.28–32 In contrast, very few synthetic allosteric regulators of TG2 activity have been identified, all of which inhibit catalytic activity.
Even though the nucleotide binding site of TG2 would be the most obvious target for allosteric inhibition, few small molecules are known to target this site, most of which are very close analogs of the natural ligands (and are, therefore, neither cell permeable nor bioavailable). This is consistent with the observation that the nucleotide binding site of TG2 is markedly different from similar sites in ATP-dependent kinases, for which a plethora of inhibitors exist that mimic nucleobase interactions.33 The sugar phosphate analog, glucosamine-6-phosphate, is a weak (millimolar) ligand for the GTP site; it appears to induce a similar conformational change as GTP itself.34
Thienopyrimidinone compounds such as the LDN-27219 analog 8 [Fig. 3(A)] are also thought to be allosteric inhibitors of TG2 activity based in their noncompetitive inhibition characteristics.35, 37 The binding site of these inhibitors has not yet been verified, and some controversy exists whether it is the GTP binding pocket itself, or a site that is coupled to the GTP binding site.35, 38 The acylidene oxindoles such as CK-IV-38 [Fig. 3(B)] comprise another class of allosteric inhibitors based on their noncompetitive inhibition characteristics. Their binding site also remains to be determined.36
Mutations and transcriptional variants
Four shorter variants of TG2 have been discovered, all resulting from alternative splicing of the transcript. One variant, transglutaminase homologue (TGH, also TGase-short) lacks the C-terminal 139 amino acids and its terminal residues 539–548 are altered from the full-length transcript.39, 40 A second variant, transglutaminase homolog 2 (TGH2) lacks the C-terminal 338 amino acids and has an altered sequence in its terminal residues 287–349.40 Two other variants (tTGv1 and tTGv2) result from alternative splicing in exons 12 and 13. They share the first 620 amino acids with full-length TG2, but have divergent 54 or 25 amino acid C-terminal sequences.41
Because TGH lacks key residues lining the nucleotide-binding pocket, it is unresponsive to GTP, as are the tTGv1 and tTGv2 variants. In each case, the protein retains some Ca-dependent transglutaminase activity.41 Upregulation of TGH has been detected under apoptotic conditions and in Alzheimer's disease patients and is believed to have a differential biological role compared with full-length TG2.42 In all likelihood, these variants have distinct calcium dependence, which could explain how TG2 is activated without the need for high Ca2+ concentration inside the cell. Although TGH2 has not yet been characterized, its sequence is significantly modified immediately following the active site Cys277, a region of the protein that includes the catalytic triad of His335 and Asp358. As such, TGH2 is likely catalytically incompetent.
A number of missense mutations in the gene encoding human TG2 have been detected and implicated in diseases such as early onset Type 2 diabetes (Fig. 4).43, 44 Three of these functionally annotated mutations map to the vicinity of the putative Ca2+ binding site S3 and have been shown to modestly reduce the transamidation activity of the enzyme, underscoring the role of Ca2+ in allosteric regulation of TG2 function.45, 46 Given the observation that TG2-null mice are developmentally and reproductively normal,47, 48 perhaps it is not surprising that mutations in the corresponding human gene have subtle, if any, phenotypes.
Figure 4. Structure of TG2 in the open (PDB 2Q3Z) conformation, colored by domain (blue: N-terminal domain, green: catalytic domain, yellow: first C-terminal β-barrel, red: second C-terminal β-barrel). The thiol of the active site Cys277 is visualized with a yellow sphere. Three positions whose mutation has been implicated with human disease (M330R, I331N, N333S, all in early-onset Type 2 diabetes) are depicted on the protein structure as brown spheres.43, 44 These mutations map to the vicinity of the putative calcium-binding site S3.45 The vicinal disulfide bond between Cys370 and Cys371 is depicted in the inset.
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