Mitochondrial Sirtuin TcSir2rp3 Affects TcSODA Activity and Oxidative Stress Response in Trypanosoma cruzi

Abstract

Trypanosoma cruzi faces a variety of environmental scenarios during its life cycle, which include changes in the redox environment that requires a fine regulation of a complex antioxidant arsenal of enzymes. Reversible posttranslational modifications, as lysine acetylation, are a fast and economical way for cells to react to environmental conditions. Recently, we found that the main antioxidant enzymes, including the mitochondrial superoxide dismutase A (TcSODA) are acetylated in T. cruzi, suggesting that protein acetylation could participate in the oxidative stress response in T. cruzi. Therefore, we investigated whether mitochondrial lysine deacetylase TcSir2rp3 was involved in the activity control of TcSODA. We observed an increased resistance to hydrogen peroxide and menadione in parasites overexpressing TcSir2rp3. Increased resistance was also found for benznidazole and nifurtimox, known to induce reactive oxidative and nitrosactive species in the parasite, associated to that a reduction in the ROS levels was observed. To better understand the way TcSir2rp3 could contributes to oxidative stress response, we analyzed the expression of TcSODA in the TcSir2rp3 overexpressing parasites and did not detect any increase in protein levels of this enzyme. However, we found that these parasites presented higher levels of superoxide dismutase activity, and also that TcSir2rp3 and TcSODA interacts in vivo. Knowing that TcSODA is acetylated at lysine residues K44 and K97, and that K97 is located at a similar region in the protein structure as K68 in human manganese superoxide dismutase (MnSOD), responsible for regulating MnSOD activity, we generated mutated versions of TcSODA at K44 and K97 and found that replacing K97 by glutamine, which mimics an acetylated lysine, negatively affects the enzyme activity in vitro. By using molecular dynamics approaches, we revealed that acetylation of K97 induces specific conformational changes in TcSODA with respect to hydrogen-bonding pattern to neighbor residues, suggesting a key participation of this residue to modulate the affinity to O2−. Taken together, our results showed for the first time the involvement of lysine acetylation in the maintenance of homeostatic redox state in trypanosomatids, contributing to the understanding of mechanisms used by T. cruzi to progress during the infection.

Beyond Histones: Unveiling the Functional Roles of Protein Acetylation in Prokaryotes and Eukaryotes

“…Previous studies have demonstrated that lysine acetylation regulates SODA activity in human and T. cruzi enzymes. Specifically, acetylation of K68 and K97 negatively affects enzyme activity in these organisms, respectively [26, 27]. To investigate whether this regulatory mechanism is conserved throughout evolution, we analyzed SODAs from A. fumigatus , D. melanogaster , D. rerio , T. cruzi , T. brucei , A. thaliana , and H. sapiens .…”

“…Notably, all SODAs possess a conserved “funnel” region that plays a crucial role in directing the substrate towards the enzyme’s active site. This region is typically enriched with positively charged lysine residues, creating a pathway for negatively charged superoxide to reach the catalytic site [26, 27]. Consequently, acetylation of any lysine residue within this region could alter substrate direction due to the neutralization of positive charges [26, 27].…”

“…Notably, all SODAs possess a conserved "funnel" region that plays a crucial role in directing the substrate towards the enzyme's active site. This region is typically enriched with positively charged lysine residues, creating a pathway for negatively charged superoxide to reach the catalytic site [26,27].…”

“…Consequently, acetylation of any lysine residue within this region could alter substrate direction due to the neutralization of positive charges [26,27].…”

Antileishmanial metallodrugs and the elucidation of new drug targets linked to post-translational modifications machinery: pitfalls and progress

“…sponse and other key cellular processes. (48,49,60,61,62) Lysine acetyltransferases (KATs) are responsible for transferring the acetyl group from acetyl-CoA to the lysine residues, while the lysine deacetylases (KDACs) remove the acetyl groups. (63,64) Moreover, bromodomain-containing proteins (BRDs) bind acetylated lysines and recruit proteins that will perform downstream regulatory functions.…”