Effect of lysine acetylation on the regulation of Trypanosoma brucei glycosomal aldolase activity

Abstract

Post-translational modifications provide suitable mechanisms for cellular adaptation to environmental changes. Lysine acetylation is one of these modifications and occurs with the addition of an acetyl group to Nε-amino chain of this residue, eliminating its positive charge. Recently, we found distinct acetylation profiles of procyclic and bloodstream forms of Trypanosoma brucei, the agent of African Trypanosomiasis. Interestingly, glycolytic enzymes were more acetylated in the procyclic, which develops in insects and uses oxidative phosphorylation to obtain energy, compared with the bloodstream form, whose main source of energy is glycolysis. Here, we investigated whether acetylation regulates the T. brucei fructose 1,6-bisphosphate aldolase. We found that aldolase activity was reduced in procyclic parasites cultivated in the absence of glucose and partial recovered by in vitro deacetylation. Similarly, acetylation of protein extracts from procyclics cultivated in glucose-rich medium, caused a reduction in the aldolase activity. In addition, aldolase acetylation levels were higher in procyclics cultivated in the absence of glucose compared with those cultivated in the presence of glucose. To further confirm the role of acetylation, lysine residues near the catalytic site were substituted by glutamine in recombinant T. brucei aldolase. These replacements, especially K157, inhibited enzymatic activity, changed the electrostatic surface potential, decrease substrate binding and modify the catalytic pocket structure of the enzyme, as predicted by in silico analysis. Taken together, these data confirm the role of acetylation in regulating the activity of an enzyme from the glycolytic pathway of T. brucei, expanding the factors responsible for regulating important pathways in this parasite.

Cyclophilin 19 secreted in the host cell cytosol byTrypanosoma cruzipromotesROSproduction required for parasite growth

“…The following primary antibodies were used at 1:20,000 dilution: anti‐Hsp70 (McDowell, Ransom, & Bangs, 1998), anti‐ T. cruzi β‐tubulin, anti‐eIF5A (Chung, Rocha, Tonelli, Castilho, & Schenkman, 2013) and anti‐BiP (Bangs, Uyetake, Brickman, Balber, & Boothroyd, 1993). Anti‐influenza virus hemagglutinin (HA.11) (Covance), anti‐aldolase (Barbosa Leite et al, 2020) and anti‐human CypA (Cell Signalling) were used at 1:5,000, anti‐GAPDH (14C10) (Cell Signalling) at 1:10,000 and CypA at 1:5,000. Anti‐p47phox (Merck, SAB4502810) were used at 1:500, and anti‐,Phospho Ser345 of p47 (PA5‐37806) from Thermo‐Fisher Scientific at 1:100.…”

Protein acetylation in the critical biological processes in protozoan parasites

“…Interestingly, fructose 1,6-biphosphate aldolase, which converts fructose 1,6-biphosphate (F-1,6-P) to dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (GA-3-P), acetylation levels are higher in procyclic forms cultivated in the absence of glucose, compared to those cultivated in the presence of glucose. This observation is associated with lower aldolase activity in procyclics grown in the absence of glucose, a phenotype that is reverted after in vitro deacetylation [81] (Figure 3B).…”

“…Human aldolase is negatively regulated by the acetylation of lysine 147 (K147) present in the catalytic site [79]. We compared T. brucei and mammalian aldolase protein structures and observed a high degree of similarity between the structures with the conservation of the residues that compose the catalytic pocket, including the regulatory K147 residue, which corresponds to K157 in T. brucei [81] (Figure 3C). T. brucei aldolase recombinant proteins mimicking an acetylated state of K157 (lysine is replaced by glutamine) abolishes the enzyme activity compared to the native form [81], similarly to mammalian aldolase [79].…”

“…Functional studies to investigate the role of acetylation on glycolytic enzymes were recently published for T. brucei and T. gondii [15,81]. The T. brucei procyclic form, which develops in the insect gut, relies on amino acids as the primary carbon source and obtains adenosine triphosphate (ATP) by oxidative phosphorylation.…”

Mitochondrial sirtuin TcSir2rp3 affects TcSODA activity and oxidative stress response in Trypanosoma cruzi

“…Interesting, in T. brucei glycolytic enzymes were found heavily acetylated and later proved to be regulated by acetylation (Barbosa Leite et al, 2020), while in T. cruzi several proteins that are central for antioxidant defense of the parasite were detected acetylated (Moretti et al, 2018).…”