Regulation of Transglutaminase 2 structure and function by Ca²⁺ and [alpha]-synuclein
Viscomi, Jessica Sofia
Transglutaminase 2 (TG2) catalyzes the Ca2+-dependent transamidation of protein substrates between glutamine and lysine residues by formation of an N[epsilon]-([gamma]-L-glutamyl)-L-lysine isopeptide bond. The Ca2+-dependent activity of this enzyme is responsible for the majority of the protein cross-linking activity in the brain associated with the early stages of pathogenic aggregate formation in neurodegenerative diseases. TG2 Ca2+-dependent activity is positively regulated by Ca2+ binding and negatively regulated by GTP binding. The prevailing view in the field is that TG2 Ca2+-dependent activity remains latent in the cell under physiological conditions arises from previously determined activation/affinity constants for Ca2+ suggest that intracellular [Ca2+] are too low to activate TG2 transamidation and intracellular [GTP] inhibit TG2 activation. However, TG2 activity and products of TG2 are detectable under normal cellular conditions in vivo; therefore, mechanisms involving sensitization to physiological [Ca2+] and de-sensitization to cellular [GTP] need to be investigated further to gain a more complete understanding of the regulation of TG2 activity. Previously determined kinetic parameters of TG2 Ca2+-dependent activity were done in the absence of physiologically relevant protein substrates of TG2 such as casein and Z-Gln-Gly (a synthetic acyl-donor substrate). In our studies, we use recombinant [alpha]-synuclein, a physiologically relevant protein substrate of TG2 in the context of AD and PD. We determined the initial rates of [alpha]-synuclein TG2-mediated polyamidation and the effect of Ca2+ on both polyamidation and GTP-[gamma]-S inhibition of TG2 transamidation activity. We also investigated the structural implications of varying Ca2+ and [alpha]-synuclein concentrations on TG2 conformation by measuring the quenching of intrinsic TG2 fluorescence. Our data shows that (1) [alpha]-synuclein reduced the required Ca2+ concentration for measurable TG2 Ca2+-dependent activity (Ka ~ 200 [mu] M) compared to Z-Gln-Gly (Ka = 2 mM), (2) [alpha]-synuclein desensitizes TG2 to inhibition by GTP in vitro and reduced the GTP-[gamma]-S inhibition constant (Ki) from 0.11 ± 0.02 to 0.21 ± 0.04 [mu]M, (3) the availability of [alpha]-synuclein reduced the Kd for TG2 Ca2+-binding at physiologically relevant concentrations of Ca2+ from 16 ± 3 to 9 ± 1 [mu]M, and (4) the presence/binding of [alpha]-synuclein de-sensitizes TG2 to GTP-[gamma]-S binding/quenching by preventing the adoption of the closed/inactive TG2 conformation. Furthermore, our data show that [alpha]-synuclein allosterically regulates TG2 Ca2+-dependent activity and Ca2+-binding, and suggest that TG2 exhibits substrate-induced activation. Altogether, our studies demonstrate the ability of a physiologically relevant protein substrate to regulate TG2 cross-linking activity by sensitizing the enzyme to low [Ca2+] and desensitize TG2 to GTP-induced inhibition. These results provide insight into potential cellular regulatory mechanisms of TG2 Ca2+-dependent activity under physiological conditions, and, further, provide a platform for future studies to investigate other potential allosteric effectors of TG2 activity including protein-binding partners and lipid membranes.
Viscomi, Jessica Sofia. (July 2016). Regulation of Transglutaminase 2 structure and function by Ca²⁺ and [alpha]-synuclein (Master's Thesis, East Carolina University). Retrieved from the Scholarship. (http://hdl.handle.net/10342/5891.)
Viscomi, Jessica Sofia. Regulation of Transglutaminase 2 structure and function by Ca²⁺ and [alpha]-synuclein. Master's Thesis. East Carolina University, July 2016. The Scholarship. http://hdl.handle.net/10342/5891. September 17, 2019.
Viscomi, Jessica Sofia, “Regulation of Transglutaminase 2 structure and function by Ca²⁺ and [alpha]-synuclein” (Master's Thesis., East Carolina University, July 2016).
Viscomi, Jessica Sofia. Regulation of Transglutaminase 2 structure and function by Ca²⁺ and [alpha]-synuclein [Master's Thesis]. Greenville, NC: East Carolina University; July 2016.
East Carolina University