Transglutaminase regulation of cell function

Abstract

Transglutaminases (TGs) are multifunctional proteins having enzymatic and scaffolding functions that participate in regulation of cell fate in a wide range of cellular systems and are implicated to have roles in development of disease. This review highlights the mechanism of action of these proteins with respect to their structure, impact on cell differentiation and survival, role in cancer development and progression, and function in signal transduction. We also discuss the mechanisms whereby TG level is controlled and how TGs control downstream targets. The studies described herein begin to clarify the physiological roles of TGs in both normal biology and disease states.

FIGURE 1.

Enzymatic reactions catalyzed by transglutaminases (TGs). Transamidation crosslinking reactions require the presence of Ca²⁺ to covalently link primary amines including polyamines, monoamines, and protein-bound amines (P2) to a glutamine residue of the acceptor protein (P1). These reactions form polyamines or monoamine crosslinks with proteins (1) or protein-protein crosslinks to form an ϵ-(γ-glutamyl)lysine isopeptide bond (2). Under slightly acidic conditions, some TGs can utilize H₂O to catalyze deamidation of the P1 protein (3).

FIGURE 2.

The TG protein catalytic sites. Amino acid sequences derived from the catalytic core of each of the nine known transglutaminases. The catalytic cysteine residue (indicated by arrow) is part of the conserved motif that is required for the transamidation reaction. This residue is replaced with alanine in the only catalytically inactive member of TGs, band 4.2 protein.

FIGURE 3.

Three TG2 conformations. Guanine nucleotide (GTP/GDP)-bound TG2 is compact (closed) and catalytically inactive. Catalytic activity refers to ability of the enzyme to perform the transamidation reaction. The structure of TG2 in its Ca²⁺-bound form has not been resolved, but a putative Ca²⁺-binding site homologous to FXIIIa is distorted by GTP/GDP binding to TG2. The binding of Ca²⁺ to the catalytic domain of TG2 alters the protein to move domains 3 and 4 away from the catalytic domain, thus making the active site accessible (open, catalytically active). Oxidation of the open/active protein results in loss of activity (open, catalytically inactive). The oxidized state can be prevented by treatment with thioredoxin. NH₂-terminal domain is blue. COOH-terminal domain is red.

FIGURE 4.

TG2-mediated adhesion/signaling at the cell surface. The solid black arrows indicate TG2-mediated activation of signaling. The dotted black line indicates binding of activated PKCα to the integrin cytoplasmic tails, causing their redistribution on the cell surface. The dashed gray arrows outline activation of syndecan-2 by intracellular PKCα and syndecan-2-mediated activation of ROCK, which induces stress fiber and focal adhesion formation. The dashed black arrow indicates nuclear translocation of β-catenin, which leads to complex formation with Tcf/Lef and activation of gene transcription. The dashed double black line indicates the unknown pathway of GPR56-induced Gα_q activation, which inhibits tumor cell growth and metastasis. The flat-headed arrows indicate inhibition of signaling.

FIGURE 5.

Regulation of signaling by TG2-induced monoaminylation. Monoamines (serotonin, norepinephrine, dopamine, etc.) interact with the monoamine receptor, but are also delivered to cells via monoamine transporters. Intracellular monoamines are covalently crosslinked to cytoplasmic proteins by TG2. Target proteins include the small regulatory GTPases (RhoA, Rac1, Rab3A, Rab4a, and Rab27A) and cytoskeletal components such as α-actin. These TG2-induced posttranslational modifications alter target protein biological activity. The biological effects of these TG2-driven modifications are important in diabetes, thrombosis, and arterial hypertension.

FIGURE 6.

TG2 expression results in constitutive activation of NF-κB via noncanonical pathway. Acute inflammation is a tightly regulated physiological process in which NF-κB is transiently activated as a result of IKK-complex mediated phosphorylation and degradation of the inhibitory protein IκBα. As IκBα is one of the downstream targets of NF-κB, its expression results in feedback inhibition of NF-κB, which limits the inflammatory response (A). In contrast, chronic inflammation is associated with constitutive activation of NF-κB owing to aberrant expression of TG2 (B). TG2 binds to IκBα resulting in its rapid degradation via a nonproteasomal pathway. Alternatively, TG2-mediated covalent crosslinking of IκBα may promote proteasomal degradation of IκBα polymers (broken arrows). TG2-activated NF-κB regulates the expression of multiple target genes that play roles in cell survival, invasion, and drug resistance. One of the TG2/NF-κB target genes is HIF-1α, a transcription factor known to promote an aggressive phenotype in cancer cells.

FIGURE 7.

TG2 GTPase activity and TG2/Ghα signaling. The GDP-TG2/Ghα-CRT/Ghβ complex is inactive. CRT is calreticulin. 1: Agonist stimulation of transmembrane G protein-coupled receptors (GPCR) induces exchange of GDP with GTP and dissociation of GTP-bound TG2/Ghα from CRT/Ghβ. 2: GTP-bound TG2/Ghα activates PLCδ1. 3/4: Signal termination occurs with GTP hydrolysis and reassociation of GDP-bound TG2/Ghα with free CRT/Ghβ. 5: PLCδ1 promotes coupling efficiency by stabilizing GTP-TG2/Ghα. 6: PLCδ1 catalyzes hydrolysis of phosphatidylinositol 4,5-bisphosphate to diacylglycerol and inositol 1,4,5-triphosphate, causing an increase in intracellular Ca²⁺ level. 7: Switching off GTPase activity of TG2/Ghα is triggered by elevated intracellular Ca²⁺.

FIGURE 8.

TG2 as a novel transcriptional regulator in the nucleus. A: TG2-mediated transcriptional regulation in patients with Huntington disease. Under normal conditions, the level of TG2 transamidation activity is low and does not interfere with transcription of key genes involved in the regulation of mitochondrial and metabolic function (e.g., PGC-1α). In Huntington disease, TG2 activity increases, leading to aminylation of histones, thereby yielding an increased net positive charge that promotes tighter packing of DNA with histones. This chromatin alteration represses target gene transcription. Reduced expression of PGC-1α and related genes contributes to the mitochondrial and metabolic dysfunction observed in Huntington disease. B: TG2-dependent enzymatic crosslinking of Sp1 transcription factor in the nucleus causes Sp1 inactivation and inhibits Sp1-mediated transcription of the prosurvival gene c-Met in hepatocytes. This transamidation-dependent mechanism mediated by nuclear TG2 is involved in liver steatohepatitis. C: TG2 binds noncovalently to c-Jun in the nucleus and prevents c-Jun/c-Fos dimerization, thereby decreasing AP1 transcription factor-dependent transcription of the MMP-9 gene in cardiomyoblasts. This nonenzymatic mechanism, mediated by nuclear TG2, is thought to be involved in ECM remodeling. D: TG2 interacts noncovalently with HIF-1β in the nucleus and prevents its dimerization with HIF-1α, thus inhibiting HIF-1 binding to the hypoxic response element (HRE) in the promoter of the Bnip3 gene leading to reduced Bnip3 transcription in neuronal cells. This nonenzymatic nuclear TG2-driven mechanism plays a role in the prosurvival effect of TG2 in stroke patients. TG indicates transamidating activity of TG2, and Ad/Sc indicates adapter/scaffolding nonenzymatic activity of TG2.

FIGURE 9.

TG2 reprograms the inflammatory signaling circuitry. Inflammatory cytokines produced by infiltrating immune cells induce TG2 expression in cancer cells and host cells (tumor-associated fibroblasts). Due to low Ca²⁺ and high GTP levels, intracellular TG2 is predominantly present as a cryptic enzyme and serves as a scaffold protein. For example, it interacts with IκBα and results in its degradation via a proteasome-independent pathway. This results in increased activation of NF-κB. Activated NF-κB translocates to the nucleus and induces expression of multiple genes. TG2 is also a target gene for NF-κB, which results in an auto-induction loop between NF-κB and TG2. NF-κB regulates genes involved in promoting drug resistance and metastasis. TG2 expression in fibroblasts induces the synthesis of ECM proteins (e.g., collagens, fibronectin). Extracellular TG2, in turn, crosslinks these and other proteins and stabilizes the ECM. Low turnover of TG2-modified ECM, coupled with enhanced synthesis of ECM component proteins, results in fibrosis and desmoplastic response, which further promote aggressive phenotype in cancer cells.

FIGURE 10.

TG2 regulates inflammatory signaling, drug resistance, and metastatic phenotype. Chronic exposure to inflammatory cytokines (produced by tumor-infiltrating immune cells) results in epigenetic regulation of TG2 and initiates the feedback cycle where TG2 activates NF-κB, and NF-κB further increases TG2 expression. HIF-1α is a downstream target of TG2-induced NF-κB. Increased expression of HIF-1α (even under normal oxygen conditions) results in activation of multiple downstream target genes, which are involved in reprogramming of cancer cells for altered metabolism, and angiogenesis. Moreover, the level of key transcription repressors (e.g., Snail, Twist, Zeb) is upregulated in TG2/NF-κB/HIF-1α-expressing cells, which leads to transdifferentiation of epithelial cells to the mesenchymal state (EMT), an important step in tumor metastasis. TG2-induced EMT promotes stem-cell traits in cancer cells and confers resistance and self-renewal ability for successful survival and growth at metastatic sites.

FIGURE 11.

Regulatory elements of human TG2 gene expression (TGM2). Glucocorticoid response element (−1399 bp), NF-κB (−1338 bp), IL-6 response element (−1190 bp), AP-2 (−634 bp), HRE (−367 bp), AP-1 (−183 bp), CAAT box (−96 bp), GC box: Sp1-binding motifs (−54 bp, −43 bp, +59 bp, and +65 bp), TATA box (−29 bp), and NF-1 (+4 bp and +12 bp). TG2 gene expression is upregulated in response to inflammation and hypoxia. Human TG2 expression is upregulated by treatment with retinoic acid, and potential retinoic acid response elements (RAR and RXR) are located in the human TG2 promoter. [From Gundemir et al. (125a), with permission from Elsevier.]

FIGURE 12.

TG2 regulation of transcription. A: TG2 expression is upregulated by inflammation. TG2 transamidates IκBα leading to NF-κB activation in response to inflammation. B: increased TG2 expression in hypoxia leads to HIF-1β dependent transcription of genes via hypoxia response element (HRE). C: TG2 suppresses PGC-1α and cyt c expression in mutant huntingtin-expressing cells. D: TG2 crosslinks Sp1 in response to ethanol treatment leading to reduced Sp1-dependent gene transcription. E: TG2 increases cAMP expression, leading to activation of CREB. [From Gundemir et al. (125a), with permission from Elsevier.]