Acyl transfer mechanisms of tissue transglutaminase

Keillor, Jeffrey W.; Clouthier, Christopher M.; Apperley, Kim Y.P.; Akbar, Abdullah; Mulani, Amina

doi:10.1016/j.bioorg.2014.06.003

Cited by 80 publications

(74 citation statements)

References 82 publications

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“…A variety of small molecular inhibitors have been described that target TG2 (22, 29, 32, 47, 50, 55). Most of these are irreversible inhibitors designed to covalently interact with the TG2 catalytic triad of the transamidase site to inhibit transamidase (crosslinking) activity (29).…”

Section: Introductionmentioning

confidence: 99%

Transamidase site-targeted agents alter the conformation of the transglutaminase cancer stem cell survival protein to reduce GTP binding activity and cancer stem cell survival

et al. 2016

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Type 2 transglutaminase (TG2) is an important cancer stem cell survival protein that exists in open and closed conformations. The major intracellular form is the closed conformation that functions as a GTP-binding GTPase and is required for cancer stem cell survival. However, at a finite rate, TG2 transitions to an open conformation that exposes the transamidase catalytic site involved in protein-protein crosslinking. The activities are mutually exclusive, as the closed conformation has GTP binding/GTPase activity, and the open conformation transamidase activity. We recently showed that GTP binding, but not transamidase activity, is required for TG2-dependent cancer stem cell invasion, migration and tumor formation. However, we were surprised that transamidase site-specific inhibitors reduce cancer stem cell survival. We now show that compounds NC9, VA4 and VA5, which react exclusively at the TG2 transamidase site, inhibit both transamidase and GTP-binding activities. Transamidase activity is inhibited by direct inhibitor binding at the transamidase site, and GTP binding is blocked because inhibitor interaction at the transamidase site locks the protein in the extended/open conformation to disorganize/inactivate the GTP binding/GTPase site. These findings suggest that transamidase site-specific inhibitors can inhibit GTP binding/signaling by driving a conformation change that disorganizes the TG2 GTP binding to reduce TG2-dependent signaling, and that drugs designed to target this site may be potent anti-cancer agents.

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Section: Introductionmentioning

confidence: 99%

Transamidase site-targeted agents alter the conformation of the transglutaminase cancer stem cell survival protein to reduce GTP binding activity and cancer stem cell survival

et al. 2016

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“…In case of human TG2 not only the hydrophobicity of the amino acids around the active site and in the substrate binding area plays a role during the reaction catalysis, but the complexity of the surrounding Trp-s (space filling property, aromatic carbon atom ring containing nitrogen) also have important effect on stabilisation of the transition states of transglutaminase activities (recently reviewed by Keillor et al 2014). Pinkas et al (2007) reported that the W332A mutation in human TG2 and the W332F mutation in rat TG2 lead to loss or lower transamidase activity, respectively (Murthy et al 2002), but in these articles only the transamidase activity was tested.…”

Section: Effect Of Changing Hydrophobic Amino Acids Around the Activementioning

confidence: 99%

Isopeptidase activity of human transglutaminase 2: disconnection from transamidation and characterization by kinetic parameters

et al. 2015

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Transglutaminase 2 (TG2) is a multifunctional protein with diverse catalytic activities and biological roles. Its best studied function is the Ca 2+ -dependent transamidase activity leading to formation of γ-glutamyl-ε-lysine isopeptide crosslinks between proteins or γ-glutamyl-amine derivatives. TG2 has a poorly studied isopeptidase activity cleaving these bonds. We have developed and characterised TG2 mutants which are significantly deficient in transamidase activity while have normal or increased isopeptidase activity (W332F) and vice versa (W278F). The W332F mutation led to significant changes of both the Km and the Vmax kinetic parameters of the isopeptidase reaction of TG2 while its calcium and GTP sensitivity was similar to the wild type enzyme. The W278F mutation resulted in six times elevated amine incorporating transamidase activity demonstrating the regulatory significance of W278 and W332 in TG2 and that mutations can change opposed activities located at the same active site. The further application of our results in cellular systems may help to understand TG2 driven physiological and pathological processes better and lead to novel therapeutic approaches where an increased amount of cross-linked proteins correlates with the manifestation of degenerative disorders.

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“…The amine donor substrate then attacks the acyl-thioester, resulting in the formation of isopeptide bond product. In the reaction, the enzymes are more stringent to the structure spanning the glutamine residues in proteins than the amine donor (Keillor et al 2014). A few non-radioactive assays for transglutaminase activity with synthetic primary amines have been used, but the extent of transamidation catalyzed by TG2 depends on the substrate affinity and the type of primary amine used (Jeon et al 2003b;McConoughey et al 2009;Stamnaes et al 2008).…”

Section: Discussionmentioning

confidence: 98%

Uncovering protein polyamination by the spermine-specific antiserum and mass spectrometric analysis

Chou

Lee

et al. 2014

Amino Acids

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The polyamines spermidine and spermine, and their precursor putrescine, have been shown to play an important role in cell migration, proliferation, and differentiation. Because of their polycationic property, polyamines are traditionally thought to be involved in DNA replication, gene expression, and protein translation. However, polyamines can also be covalently conjugated to proteins by transglutaminase 2 (TG2). This modification leads to an increase in positive charge in the polyamine-incorporated region which significantly alters the structure of proteins. It is anticipated that protein polyamine conjugation may affect the protein-protein interaction, protein localization, and protein function of the TG2 substrates. In order to investigate the roles of polyamine modification, we synthesized a spermine-conjugated antigen and generated an antiserum against spermine. In vitro TG2-catalyzed spermine incorporation assays were carried out to show that actin, tubulins, heat shock protein 70 and five types of histone proteins were modified with spermine, and modification sites were also identified by liquid chromatography and linear ion trap-orbitrap hybrid mass spectrometry. Subsequent mass spectrometry-based shotgun proteomic analysis also identified 254 polyaminated sites in 233 proteins from the HeLa cell lysate catalyzed by human TG2 with spermine, thus allowing, for the first time, a global appraisal of site-specific protein polyamination. Global analysis of mouse tissues showed that this modification really exists in vivo. Importantly, we have demonstrated that there is a new histone modification, polyamination, in cells. However, the functional significance of histone polyamination demands further investigations.

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Acyl transfer mechanisms of tissue transglutaminase

Cited by 80 publications

References 82 publications

Transamidase site-targeted agents alter the conformation of the transglutaminase cancer stem cell survival protein to reduce GTP binding activity and cancer stem cell survival

Transamidase site-targeted agents alter the conformation of the transglutaminase cancer stem cell survival protein to reduce GTP binding activity and cancer stem cell survival

Isopeptidase activity of human transglutaminase 2: disconnection from transamidation and characterization by kinetic parameters

Uncovering protein polyamination by the spermine-specific antiserum and mass spectrometric analysis

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