The diamond anniversary of tissue transglutaminase: a protein of many talents

Abstract

Tissue transglutaminase (tTG) is capable of binding and hydrolyzing GTP, as well as catalyzing an enzymatic transamidation reaction that crosslinks primary amines to glutamine residues. tTG adopts two vastly different conformations, depending on whether it is functioning as a GTP-binding protein or a crosslinking enzyme. It has been shown to have important roles in several different aspects of cancer progression, making it an attractive target for therapeutic intervention. Here, we highlight many of the major findings involving tTG since its discovery 60 years ago, and describe recent drug discovery efforts that target specific activities or conformations of this unique protein.

Figure 1

A timeline of major discoveries and accomplishments related to tTG. Events are classified as related to overall structure and mechanism (solid black outline), general biological function (dashed black outline) or disease states (dotted black outline).

Figure 2

Catalysis of the tTG transamidation reaction. (a) The catalytic crosslinking site of tTG, from PDB ID 2Q3Z. The catalytic triad is shown in orange, whereas the transition state stabilizing Trp241 is shown in yellow. A loop formed by residues 329–334 is hidden for clarity. (b) The four catalytic residues are conserved between the eight catalytically active transglutaminase family members (alignment on the right, family members other than tTG are designated by gene symbol). Band 4.2 (EPB42), which has an alanine residue at position 277, is deficient in crosslinking activity. (c) Two proposed mechanisms for transamidation catalysis by tTG. These differ primarily by whether Trp241 serves to stabilize a cysteine–glutamine adduct via H-bonding (right path), or if it turns glutamine into a better electrophile via a cation–pi interaction (left path, cation–pi interaction shown as red double-dotted line).

Figure 3

Structure and conformations of tTG. (a) The tertiary structure of tTG. tTG adopts a ‘closed’ crosslinking inactive state (left, PDB ID 1KV3) and an ‘open’ crosslinking capable state (right, PDB ID 2Q3Z). The conformation that tTG adopts is controlled by the addition of either calcium or guanine nucleotides. The different domains in tTG are indicated by color: the N-terminal sandwich is red, the crosslinking catalytic domain is blue and the subsequent two β sheets are green and cyan. Panels b–e use the same coloring. (b) The nucleotide binding site of closed-state tTG (1KV3). The binding site is comprised nearly equally by residues from the crosslinking catalytic domain and the first β-sheet domain. GDP is shown in orange. (c) The nucleotide binding site of open-state tTG (2Q3Z). The β sheets move away from the catalytic domain, and insufficient residues remain to provide a binding pocket for the nucleotide. GDP, in orange, is overlayed from 1KV3. (d) The crosslinking substrate-binding site of closed-state tTG (1KV3). A substrate-mimetic inhibitor from 2Q3Z was overlaid onto 1KV3. Although the binding site exists in the crosslinking catalytic domain, it is occupied by residues from the β sheets. (e) The crosslinking substrate-binding site of open-state tTG (2Q3Z). A substrate-mimetic inhibitor (orange) fits into a groove in the crosslinking catalytic site. (f) Overlay of GDP and GTP bound tTG structures (1KV3 and 4PYG, respectively). The GDP-bound structure is colored gray, and GDP is shown as gray spheres. The GTP-bound structure is colored by B-factor, where shades of blue are stable, immobile residues and shades of orange are flexible, poorly defined residues. The regions of the protein that have poor overlap between the GDP- and GTP-bound structures, indicated by red arrows, are naturally highly mobile in the crystal structures. (g) Zoomed in view of the lower indicated loop in (f).

Figure 4

tTG is highly expressed in aggressive cancers. Mean survival time in cancers (top [50]) correlates to mRNA expression of the TGM2 gene (middle, images are representative sections of The Cancer Genome Atlas data [52] prepared with the UCSF Cancer browser [51]) and to tTG protein expression (as determined by staining of tissue slices with the tTG-specific antibody CAB002598 at http://www.proteinatlas.org). mRNA and protein expression for tumor samples or average healthy tissue are shown in similar color schemes, with red representing high expression, white moderate expression, blue low expression and gray no detectable expression.

Figure 5

tTG regulates many signaling events that promote cancer cell phenotypes. The signaling pathways most often mutated in pancreatic cancers are shown in blue, whereas the phenotypes shared by most cancers are shown in purple. Most of the signaling pathways increase tTG expression, whereas positive feedback mechanisms allow tTG to enhance signaling in several such pathways. Of the phenotypes identified as hallmarks of cancer, tTG has demonstrated roles in about half of them.

Figure 6

Inhibitors of tTG. The most common inhibitors of tTG are: (a) pseudo-substrate inhibitors, such as MDC and cystamine, which replace the amine-bearing substrate in tTG catalyzed crosslinking reactions; and (b) peptidomimetic inhibitors bearing ‘warhead’ functionality that form covalent bonds to the catalytic Cys277 and irreversibly inhibit tTG crosslinking. For peptidomimetic compounds, the warhead functionalities are shown in red. K_i or IC₅₀ values against tTG crosslinking in vitro are presented for each compound in (b) only because pseudo-substrates do not inhibit crosslinking, whereas all compounds have a GI₅₀ value against cell culture systems reported. References are provided to manuscripts providing the related data. ^aData taken from http://www.zedira.com/resources/content/pdf/zedira_poster_oslo_2011.pdf.

Figure 7

Novel scaffolds of tTG inhibitors. A wide variety of inhibitors of tTG have been reported that are neither pseudo-substrates or peptidomimetics. Some are irreversible, but most are reversible inhibitors. IC₅₀ values for tTG in vitro are reported, as are cell growth GI₅₀ values where appropriate, along with the related references.