Toxoplasma gondii UBL‐UBA shuttle proteins contribute to the degradation of ubiquitinylated proteins and are important for synchronous cell division and virulence

Abstract

Toxoplasma gondii is an obligate intracellular apicomplexan parasite that causes lethal diseases in immunocompromised patients. Ubiquitin‐proteasome system (UPS) regulates many cellular processes by degrading ubiquitinylated proteins. The UBL‐UBA shuttle protein family, which escorts the ubiquitinylated proteins to the proteasome for degradation, are crucial components of UPS. Here, we identified three UBL‐UBA shuttle proteins (TGGT1_304680, DNA damage inducible protein 1, DDI1; TGGT1_295340, UV excision repair protein rad23 protein, RAD23; and TGGT1_223680, ubiquitin family protein, DSK2) localized in the cytoplasm and nucleus of T gondii. Deletion of shuttle proteins inhibited parasites growth and resulted in accumulation of ubiquitinylated proteins. Cell division of triple‐gene knockout strain was asynchronous. In addition, we found that the retroviral aspartic protease activity of the nonclassical shuttle protein DDI1 was important for the virulence of Toxoplasma in mice. These results showed the critical roles of UBL‐UBA shuttle proteins in regulating the degradation of ubiquitinylated proteins and cell division of T gondii.

The Aspartyl Protease Ddi1 Is Essential for Erythrocyte Invasion by the Malaria Parasite

“…While the presence or absence of UBL or UBA domains varies between different eukaryotic Ddi1 proteins, the RVP domain remains well conserved, suggesting a conserved catalytic role. As in budding yeast (Yip et al, 2020), humans cells (Dirac- Svejstrup et al, 2020) and Toxoplasma gondii (H. Zhang et al, 2020), we find that loss of PfDdi1 results in the accumulation of ubiquitylated proteins, which are of high molecular weight, almost certainly indicating that they contain very long ubiquitin chains (Dirac- Svejstrup et al, 2020;Yip et al, 2020). Importantly, parasites that are reliant on mutant PfDdi1 also show a build-up of these hyper-ubiquitinated species.…”

“…Whether Plasmodium Ddi1 (PfDdi1) shares conserved functions with its human and yeast homologues is unknown. Interestingly, a very recent study looked at the role of Ddi1 in the closely related apicomplexan parasite Toxoplasma gondii (H. Zhang et al, 2020). Although KO of TgDdi1 had no effect in parasite replication in vitro, its KO leads to an accumulation of ubiquitinated proteins.…”

“…Although KO of TgDdi1 had no effect in parasite replication in vitro, its KO leads to an accumulation of ubiquitinated proteins. Importantly, TgDdi1KO parasites were less virulent in vivo, a phenotype than could be rescued through complementation with PfDdi1 (H. Zhang et al, 2020). In addition to the RVP domain, PfDdi1 contains an N-terminal UBL but lacks C-terminal UBA or UIM domains that can be found in ScDdi1, TgDdi1 or hDdi2.…”

“…, humans cells (Dirac-Svejstrup et al, 2020) and Toxoplasma gondii (H Zhang et al, 2020),. we find that loss of PfDdi1 results in the accumulation of ubiquitylated proteins, which are of high molecular weight, almost certainly indicating that they contain very long ubiquitin chains (Dirac-Svejstrup et al, 2020;Yip et al, 2020).…”

Artemisinin acts by inhibiting Plasmodium falciparum Ddi1, a retropepsin, resulting into the accumulation of ubiquitinated proteins

“…PfDdi1, an essential retropepsin in the UPS 20,21 , has been shown to compensate for proteasome dysfunction and its knock out leads to polyubiquitination of proteins in both yeast and Toxoplasma gondii cells [22][23][24] . It is feasible, therefore, to speculate that artemisinin might be compromising the activity of PfDdi1 in restoring protein homeostasis following the damage.…”