The Different Conformational States of Tissue Transglutaminase Have Opposing Affects on Cell Viability

Abstract

Tissue transglutaminase (tTG) is an acyltransferase/GTP-binding protein that contributes to the development of various diseases. In human cancer cells, tTG activates signaling pathways that promote cell growth and survival, whereas in other disorders (i.e. neurodegeneration), overexpression of tTG enhances cell death. Therefore, it is important to understand how tTG is differentially regulated and functioning to promote diametrically distinct cellular outcomes. Previous structural studies revealed that tTG adopts either a nucleotide-bound closed conformation or a transamidation-competent open conformation. Here we provide evidence showing that these different conformational states determine whether tTG promotes, or is detrimental to, cell survival, with the open conformation of the protein being responsible for inducing cell death. First, we demonstrate that a nucleotide binding-defective form of tTG, which has previously been shown to induce cell death, assumes an open conformation in solution as assessed by an enhanced sensitivity to trypsin digestion and by small angle x-ray scattering (SAXS) analysis. We next identify two pairs of intramolecular hydrogen bonds that, based on existing x-ray structures, are predicted to form between the most C-terminal β-barrel domain and the catalytic core domain of tTG. By disrupting these hydrogen bonds, we are able to generate forms of tTG that constitutively assume an open conformation and induce apoptosis. These findings provide important insights into how tTG participates in the pathogenesis of neurodegenerative diseases, particularly with regard to the actions of a C-terminal truncated form of tTG (TG-Short) that has been linked to such disorders and induces apoptosis by assuming an open-like conformation.

Keywords: apoptosis; cell death; conformational change; protein cross-linking; small-angle x-ray scattering (SAXS); transglutaminase.

FIGURE 1.

tTG adopts two different conformational states. A, linear representations of tTG WT (top) and tTG-Short (bottom). tTG WT is composed of four domains including an N-terminal β-sandwich (blue) and a catalytic core (green) followed by β-barrel 1 (orange) and β-barrel 2 (red) at its C terminus. tTG-Short is an alternative splice variant of tTG that lacks approximately the last third of β-barrel 1 and all of β-barrel 2. The numbers indicate the amino acids that encode each domain. B, the x-ray crystal structures and simplified diagrams show the closed (catalytically inactive) and open (catalytically active) conformations of tTG WT as well as the open-like conformation that tTG-Short is suspected to assume. The x-ray crystal structure of the closed conformation of tTG WT (far left) represents the GDP-bound form of the protein (PDB code 1KV3), whereas the structure shown for the open state conformation (center) represents tTG WT covalently modified with a substrate-mimetic gluten peptide (PDB code 2Q3Z). GDP and the gluten peptide are shown in sticks and colored in blue and red, respectively. The simplified diagrams show how the different domains in tTG WT are arranged in the closed (far left) versus open (middle) conformations as well as are used to highlight the suspected conformation of tTG-Short (far right). The various domains in tTG are labeled as follows; the N-terminal β-sandwich (β-sand.), the catalytic core (Core), and the two C-terminal β-barrels (β1 and β2).

FIGURE 2.

tTG WT and tTG-Short differently influence cell survival. A, cell lysates collected from NIH3T3 fibroblasts that were either treated with RA for the indicated lengths of time (left panels) or ectopically expressing the vector alone, Myc-tagged tTG WT, or Myc-tagged tTG-Short for ∼36 h (right panels) were immunoblotted (IB) with tTG and actin antibodies. B, cell lysates collected from NIH3T3 fibroblasts ectopically expressing either the vector alone for 36 h or Myc-tagged forms of either tTG WT and tTG-Short for the indicated lengths of time were immunoblotted with Myc and actin antibodies. C, NIH3T3 cells ectopically expressing the vector alone, Myc-tagged tTG WT, and Myc-tagged tTG-Short were either cultured in medium containing 2% CS or serum-starved (SS) for ∼36 h and fixed. Immunofluorescence (IF) was performed on the cells using a Myc antibody to detect the transfectants. The cells were also stained with DAPI to label nuclei. Representative images of the transfectants and their nuclei are shown. Arrows highlight the nuclei of a given transfectant, whereas asterisks indicate condensed/blebbed (apoptotic) nuclei. D, the experiment shown in C was performed three separate times, and the percent apoptosis for each condition was determined by calculating the ratio of apoptotic to non-apoptotic cells. The data shown represent the means ± S.E. E, cell lysates collected from NIH3T3 cells ectopically expressing either the vector alone for 48 h or the indicated combinations of Myc-tagged forms of tTG WT and tTG-Short for 24 and 48 h were immunoblotted with Myc and actin antibodies. F, cells ectopically expressing the vector alone, Myc-tagged tTG WT, HA-tagged tTG-Short, or Myc-tagged tTG WT and HA-tagged tTG-Short were cultured in medium containing 2% CS for ∼36 h and fixed. Immunofluorescence was performed on the cells using Myc and HA antibodies to detect the transfectants. The cells were also stained with DAPI to label nuclei. The percent of cells with condensed/blebbed (apoptotic) nuclei for each condition was determined by calculating the ratio of apoptotic to non-apoptotic cells. The experiment was performed three separate times, and the data shown represent mean ± S.E.

FIGURE 3.

GTP binding-defective forms of tTG induce cell death. A, the indicated recombinant forms of tTG were expressed, purified, and then resolved by SDS-PAGE. The gels were stained with Coomassie Blue to visualize the proteins. A protein molecular mass (M.W.) marker was included on the gel to show the sizes of the recombinant proteins. B, the recombinant tTG proteins (600 nm) were subjected to fluorescent measurements using a spectrofluorimeter. BODIPY FL GTPγS was added to the reactions (labeled on the graph as BODIPY-GTP Added), and changes in fluorescence were determined. At the indicated times, an excess of unlabeled GTP was added to the reactions (labeled on the graph as GTP Added). These experiments were performed at least three separate times, each yielding similar results. C, cell lysates collected from NIH3T3 fibroblasts ectopically expressing the indicated forms of Myc-tagged tTG were assayed for their enzymatic transamidation activity as read out by the incorporation of BPA into cell lysate proteins (top panel). The same lysates were also immunoblotted (IB) with Myc and actin antibodies (bottom two panels). D, cell lysates collected from NIH3T3 fibroblasts ectopically expressing the vector-alone for 36 h and either Myc-tagged tTG WT or Myc-tagged tTG R580K for the indicated lengths of time were immunoblotted with Myc and actin antibodies. E, NIH3T3 cells ectopically expressing the vector alone or one of the indicated Myc-tagged tTG proteins were cultured in medium containing 10% CS for ∼36 h and fixed. Immunofluorescence was performed on the cells using a Myc antibody to detect the transfectants, and the cells were also stained with DAPI to label nuclei. Cells undergoing apoptosis were identified by nuclear condensation/blebbing. The experiment was performed three separate times, and the percent apoptosis for each condition was determined by calculating the ratio of apoptotic to non-apoptotic cells. The data shown represent the mean ± S.E. F, NIH3T3 cells ectopically expressing the vector alone or one of the indicated Myc-tagged tTG proteins were cultured in medium containing 2% CS without (−) or with either monodansylcadaverine or Z-DON for ∼36 h and fixed. Immunofluorescence was performed on the cells using a Myc antibody to detect the transfectants, and the cells were also stained with DAPI to label nuclei. Cells undergoing apoptosis were identified by nuclear condensation/blebbing. The experiment was performed three separate times, and the percent apoptosis for each condition was determined by calculating the ratio of apoptotic to non-apoptotic cells. The data shown represent mean ± S.E.

FIGURE 4.

tTG R580K and tTG-Short adopt open conformations in solution. A, purified recombinant forms of tTG WT and tTG R580K (3.5 μg of each) were incubated without or with trypsin for 2 h before being resolved by SDS-PAGE and stained with Coomassie Blue to visualize the proteins. Note that the tTG R580K mutant is more susceptible to trypsin digestion than tTG WT. B, SAXS analysis was performed on recombinant tTG WT. A monomeric model derived from the crystal structure of GDP-bound wild type tTG (PDB code 1KV3) was fitted into the calculated SAXS envelope, with the N-terminal β-sandwich and the catalytic core domains colored in blue and the two C-terminal β-barrel domains colored in red (left panel). A schematic representation shows the superimposition of the fitted model (blue and red) onto the structure of wild type tTG bound to GDP (PDB code 1KV3, yellow) (middle panel). The experimental scattering profiles from SAXS are shown as blue dots, and the scattering profile for the GDP-bound wild type tTG structure (PDB code 1KV3) is shown as a green line (right panel). C, SAXS analysis was performed on recombinant tTG R580K. Two monomeric models derived from a substrate-bound tTG WT crystal structure (PDB code 2Q3Z) were fitted into the calculated SAXS envelope for tTG R580K in a head-to-tail fashion. The N-terminal β-sandwich and the catalytic core domains of the fitted models are colored in blue, and the two C-terminal β-barrel domains are colored in red (left panel). A schematic representation shows the superimposition of one of the monomers in the fitted model (blue and red) onto the structure of the substrate-bound tTG (PDB code 2Q3Z, yellow) (middle panel). The experimental scattering profiles from SAXS are shown as blue dots, and the scattering profile for the substrate-bound tTG WT structure (PDB code 2Q3Z) is shown as a green line (right panel). D, SAXS analysis was performed on recombinant tTG-Short. Two monomeric models derived from a substrate-bound tTG WT crystal structure (PDB code 2Q3Z) were fitted into the calculated SAXS envelope for tTG-Short in a head-to-tail fashion. One monomer is colored in red and the other in blue (left panel). A schematic representation shows one of the monomers in the fitted model. The N-terminal β-sandwich is colored in blue, and the catalytic core domain and the portion of β-barrel that is present in tTG-Short are colored red (middle panel). The experimental scattering profiles from SAXS are shown as blue dots, and the scattering profile for the substrate-bound tTG WT structure (PDB code 2Q3Z) is shown as a green line (right panel).

FIGURE 5.

Identification of a region in the C terminus of tTG that is crucial for its ability to assume the closed conformation. Linear representations showing the relative lengths of Myc-tagged forms of tTG WT, tTG-Short, and a tTG 1–672. The numbers shown indicate amino acids. B, cell lysates collected from NIH3T3 fibroblasts ectopically expressing the vector alone or one of the indicated Myc-tagged tTG proteins for ∼24 h were immunoblotted (IB) with Myc and actin antibodies. C, NIH3T3 cells ectopically expressing the vector alone or one of the indicated Myc-tagged tTG proteins were cultured in medium containing 10% CS for ∼36 h and fixed. Immunofluorescence was performed on the cells using a Myc antibody to detect the transfectants, and the cells were also stained with DAPI to label nuclei. Cells undergoing apoptosis were identified by nuclear condensation/blebbing. The experiment was performed three separate times, and the percent apoptosis for each condition was determined by calculating the ratio of apoptotic to non-apoptotic cells. The data shown represent the mean ± S.E. D, the C-terminal 15 amino acids of tTG (residues 673–687) are highlighted in purple in the x-ray crystal structure of tTG bound to GDP (top panel). Two pairs of hydrogen bonds formed between residues Asn-681 and Asp-434 and between residues Lys-677 and Trp-254 are shown in magenta with participating residues shown in sticks (bottom panels). The distances of each interaction are shown.

FIGURE 6.

The identification of key residues in tTG that are essential for its ability to adopt the closed conformation. A, cell lysates collected from NIH3T3 cells ectopically expressing the indicated forms of Myc-tagged tTG were assayed for their enzymatic transamidation activity (top panel) as well as immunoblotted (IB) with Myc and actin antibodies (bottom two panels). The lines indicate where a portion of the blot was removed. B, the indicated recombinant forms of tTG were expressed, purified, and then resolved by SDS-PAGE. The gels were stained with Coomassie Blue to visualize the proteins. A protein molecular mass (M.W.) marker was included on the gel to show the sizes of the recombinant proteins. C, purified recombinant forms of tTG WT, tTG W254A, and tTG N681A (600 nm) were incubated with BODIPY-GTP (indicated on the graph as BODIPY-GTP Added), and the resulting changes in fluorescence were determined using a spectrofluorimeter. These experiments were performed at least three separate times, each yielding similar results. D, purified recombinant forms of tTG WT, tTG R580K, tTG W254A, and tTG N681A (3.5 μg of each) were incubated without or with trypsin for 2 h before being resolved by SDS-PAGE and then stained with Coomassie Blue to visualize the proteins. Note that only tTG WT was not efficiently digested by trypsin. E, SAXS was performed on purified recombinant tTG W254A. Two monomeric models derived from a substrate-bound tTG WT crystal structure (PDB code 2Q3Z) were fitted into the calculated SAXS envelope for tTG-Short in a head-to-tail fashion. The N-terminal β-sandwich and the catalytic core domains of the fitted models are colored in blue, and the two C-terminal β-barrel domains are colored in red (left panel). A schematic representation shows the superimposition of one of the monomers in the fitted model (blue and red) (right panel). The experimental scattering profiles from SAXS are shown as blue dots, and the scattering profile for the substrate-bound tTG WT structure (PDB code 2Q3Z) is shown as a green line (bottom panel).

FIGURE 7.

Forms of tTG that assume an open conformation are consistently cytotoxic. A, cell lysates collected from NIH3T3 cells ectopically expressing the vector alone or the indicated forms of Myc-tagged tTG were immunoblotted (IB) with Myc and actin antibodies. B, NIH3T3 fibroblasts ectopically expressing the same Myc-tagged tTG proteins shown in A were cultured in medium containing 10% CS for ∼36 h and fixed. Immunofluorescence was performed on the cells using a Myc antibody to detect the transfectants. The cells were also stained with DAPI to label nuclei. Apoptotic cells were identified by nuclear condensation/blebbing. The experiment was performed three separate times, and the percent apoptosis for each condition was determined by calculating the ratio of apoptotic to non-apoptotic cells. The data shown represent the mean ± S.E.

FIGURE 8.

Diagram highlighting the different conformational states of tTG and the effects they have on cells. tTG WT primarily adopts a closed conformation in cells due to the relatively high levels of GTP or GDP and low levels of Ca²⁺ (left panel). Under these conditions tTG has been shown to promote cell growth and survival. However, in response to cell stresses, intracellular Ca²⁺ levels can be increased, resulting in less GTP/GDP binding to tTG and allowing for it to adopt an open, catalytically active conformation (middle panel). If tTG persists in the open conformation for extended lengths of time, it induces cell death. Interestingly, tTG-Short is a splice variant of tTG that lacks the portion of the C terminus determined to be important for keeping it in the closed conformation. Consistent with this finding, tTG-Short expression in cells induces apoptosis (right panel).