Varma A and Storey KB (2023), Hepatic citrate synthase suppression in the fre...

XB-ART-59940

Int J Biol Macromol 2023 Jul 01;242Pt 1:124718. doi: 10.1016/j.ijbiomac.2023.124718.

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Hepatic citrate synthase suppression in the freeze-tolerant wood frog (Rana sylvatica).

Varma A , Storey KB .

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The wood frog, Rana sylvatica endures whole body freezing for weeks/months while overwintering at subzero temperatures. Survival of long-term freezing requires not only cryoprotectants but also strong metabolic rate depression (MRD) and reorganization of essential processes in order to maintain a balance between ATP-producing and ATP-consuming processes. Citrate synthase (CS) (E.C. 2.3.3.1) is an important irreversible enzyme of the tricarboxylic acid (TCA) cycle and forms a crucial checkpoint for many metabolic processes. Present study investigated the regulation of CS from wood frog liver during freezing. CS was purified to homogeneity by a two-step chromatographic process. Kinetic and regulatory parameters of the enzyme were investigated and, notably, demonstrated a significant decrease in the Vmax of the purified form of CS from frozen frogs as compared to controls when assayed at both 22 °C and 5 °C. This was further supported by a decrease in the maximum activity of CS from liver of frozen frogs. Immunoblotting also showed changes in posttranslational modifications with a significant decrease in threonine phosphorylation (by 49 %) for CS from frozen frogs. Taken together, these results suggest that CS is suppressed and TCA flux is inhibited during freezing, likely to support MRD survival of harsh winters.

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GO keywords: tricarboxylic acid cycle [+]

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	Fig. 1. Purification of citrate synthase (CS) from liver of control wood frogs (Rana sylvatica) separated via SDS-PAGE with a 10 % acrylamide gel and visualized with silver staining. Lanes are as follows: 1: Molecular weight ladder, 2: Purified CS from the wood frog liver, 3: Crude homogenate, 4: Commercially purified CS from porcine heart (Sigma-Aldrich #C3260).
	Fig. 2. Comparison of CS activity in whole liver extracts from control (unfrozen) versus frozen frogs, expressed as mU activity per mg total protein, mean ± SEM, n ≥ 4 independent trials. *-Significantly different from the control value as assessed by Student's t-test, p < 0.05.
	Fig. 3. Western blot analysis assessing differences in posttranslational modifications of R. sylvatica liver CS between control (unfrozen) and frozen conditions. Relative band intensity refers to the quotient of the intensity of the chemiluminescent signal divided by the band intensity observed after staining with Coomassie brilliant blue. Data are mean ± SEM, n ≥ 4 independent trials. *-Significantly different from the control value as assessed by Student's t-test, p < 0.05.
	Fig. 4. Predicted threonine phosphorylation sites as determined from the CS sequence of R. temporaria by Netphos 3.1. (A) Predicted threonine residues (blue), and predicted threonine residues that can likely be phosphorylated are shown in yellow color and labeled according to position in the sequence. (B) Diagram depicting calculated scores for each threonine residue in relation to the threshold line (shown in red). Threonine residues with a score above threshold (>0.500) are predicted to be probable phosphorylation sites. (C) Threonine residues above the threshold and the protein kinases that are predicted to be able to phosphorylate them.