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We recently applied carbonic anhydrase (CA) for the rapid catalytic conversion of carbon dioxide to enable the self-healing properties of concrete and in the development of a carbon-negative concrete replacement named Enzymatic Construction Material (ECM). Here, we explore the stability and carbonate generation ability of model molecular mimics of carbonic anhydrase under high pH and elevated temperatures relevant to long-term durability in cementitious and concrete-like materials. Molecular mimics include Zn2+-based organometallic complexes with an aromatic ligand tris(2-pyridylmethyl)amine, TPA, and with an aliphatic ligand cyclen, 1,4,7,10-tetraazacyclododecane. The Zn(TPA) and Zn(cyclen) complexes are stable in aqueous environments at standard pressures ranging from neutral to pH 13 and temperatures up to 120 °C, where CA is inactive. Under the temperature and pH conditions studied, organometallic degradation pathways do not involve the decomposition of either organic ligand but rather the dissociation of the complex that is reversible upon neutralization in the case of Zn(TPA). Zn(cyclen) is stable at high temperatures at pH 12 and above, resembling cementitious conditions for over 365 days with no signs of degradation. Separately, alkaline calcium-containing solutions with either 25 nM CA or 5 mM Zn(cyclen) catalyst demonstrated accelerated pH decreases compared to catalyst-free controls upon sparging with carbon dioxide because of the conversion of CO2 and H2O to HCO3– and H+. Notably, the inclusion of sub-molar concentrations of detergents, such as sodium dodecyl sulfate, in carbonate production reactions demonstrated no change in the reactivity of control solutions or those with the Zn(cyclen) catalyst but severely attenuated the conversion in CA-containing solutions concomitant with CA denaturation and loss of enzymatic activity. 

Abstract The Gαq/phospholipase Cβ1 (PLCβ1) signaling system mediates calcium responses from hormones and neurotransmitters. While PLCβ1 functions on the plasma membrane, there is an atypical cytosolic population that binds Argonaute 2 (Ago2) and other proteins associated with stress granules preventing their aggregation. Activation of Gαq relocalizes cytosolic PLCβ1 to the membrane, releasing bound proteins, promoting the formation of stress granules. Here, we have characterized Ago2 stress granules associated with Gαq activation in differentiated PC12 cells, which have a robust Gαq/PLCβ1 signaling system. Characterization of Ago2-associated stress granules shows shifts in protein composition when cells are stimulated with a Gαq agonist, or subjected to heat shock or osmotic stress, consistent with the idea that different stresses result in unique stress granules. Purified Ago2 stress granules from control cells do not contain RNA, while those from heat shock contain many different mRNAs and miRs. Surprisingly, Ago2 particles from cells where Gαq was stimulated show only two transcripts, chromogranin B, which is involved in secretory function, and ATP synthase 5f1b, which is required for ATP synthesis. RT-PCR, western blotting and other studies support the idea that Gαq-activation protects these transcripts. Taken together, these studies show a novel pathway where Gαq/PLCβ regulates the translation of specific proteins.