A comparative in-silico analysis of autophagy proteins in ciliates

Abstract

Autophagy serves as a turnover mechanism for the recycling of redundant and/or damaged macromolecules present in eukaryotic cells to re-use them under starvation conditions via a double-membrane structure known as autophagosome. A set of eukaryotic genes called autophagy-related genes (ATGs) orchestrate this highly elaborative process. The existence of these genes and the role they play in different eukaryotes are well-characterized. However, little is known of their role in some eukaryotes such as ciliates. Here, we report the computational analyses of ATG genes in five ciliate genomes to understand their diversity. Our results show that Oxytricha trifallax is the sole ciliate which has a conserved Atg12 conjugation system (Atg5-Atg12-Atg16). Interestingly, Oxytricha Atg16 protein includes WD repeats in addition to its N-terminal Atg16 domain as is the case in multicellular organisms. Additionally, phylogenetic analyses revealed that E2-like conjugating protein Atg10 is only present in Tetrahymena thermophila. We fail to find critical autophagy components Atg5, Atg7 and Atg8 in the parasitic ciliate Ichthyophthirius multifiliis. Contrary to previous reports, we also find that ciliate genomes do not encode typical Atg1 since all the candidate sequences lack an Atg1-specific C-terminal domain which is essential for Atg1 complex formation. Consistent with the absence of Atg1, ciliates also lack other members of the Atg1 complex. However, the presence of Atg6 in all ciliates examined here may rise the possibility that autophagosome formation could be operated through Atg6 in ciliates, since Atg6 has been shown as an alternative autophagy inducer. In conclusion, our results highlight that Atg proteins are partially conserved in ciliates. This may provide a better understanding for the autophagic destruction of the parental macronucleus, a developmental process also known as programmed nuclear death in ciliates.

The evolution of autophagy proteins – diversification in eukaryotes and potential ancestors in prokaryotes

“…The MIT domains in Atg1, ULK1 and ULK2 interact with Atg13/ATG13 and play an important role in autophagy (Caballe et al, 2015;Nishimura and Tooze, 2020). However, ( putative) Atg1 homologs that do not possess tandem MIT domains have also been identified, including ULK4 and STK36/Fused in metazoa, plants and protists (Dictyostelium, an amoeba species, also has a STK36/Fused homolog, TsuA) (Maloverjan and Piirsoo, 2012;Oh et al, 2005;Préat et al, 1990;Preuss et al, 2020;Tang et al, 2008), Atg1t in seeding plants (Huang et al, 2019;Suttangkakul et al, 2011) and almost all Alveolata homologs (Aslan et al, 2017;Földvári-Nagy et al, 2015) (Fig. 3A).…”

The Autophagy Machinery in Human-Parasitic Protists; Diverse Functions for Universally Conserved Proteins

“…H. sapiens has ATG101 instead of ATG29 and ATG31. Conservation and function of ATGs in Dictyostelium discoideum are reviewed in [23,24], in [25] for Chlamydomonas reinhardtii (green algae) and Cyanidioschyzon merolae (red algae), and in [26] for Tetrahymena thermophila. Additional searches for members of the poorly conserved initiation complex, ATG11/FIP200, ATG17, and ATG29/ATG31 (ATG101), were conducted for C. reinhardtii, C. merolae, and T. thermophila, using human or yeast homologs as queries.…”

“…Here, we provide an updated overview of the conservation of ATGs in parasitic protists (Figure 3B), which has been summarized in several previously published reviews [43][44][45]. Additionally, updated reviews have been published for specific protist lineages, whether they are parasitic like Apicomplexa [46,47], or free-living like photosynthetic algae [25], Ciliates [26], and Amoebozoa (Dictyostelium discoideum) [24]. Overall, the presence of partially-conserved molecular machinery in these very phylogenetically diverse eukaryotes, suggests parts of the machinery may have been present already in the last eukaryotic common ancestor (LECA) [48].…”

“…The pathogenic amoeba Entamoeba, for instance, has probably lost the complex, since its close phylogenetic relative Dictyostelium conserves all components of the ATG1 complex. ATG9 is conserved in Trypanosomatidae and Amoebozoa, but only partially conserved in Apicomplexa (it is only present in Coccidia, like Toxoplasma for instance), and is seemingly absent from Ciliates [26].…”

Ultrastructure of vegetative cells and resting cysts, and live observations of the encystation and excystation processes in Diophrys oligothrix Borror, 1965 (Protista, Ciliophora)

“…When environmental conditions are suitable again, excystment occurs, and they return to an active state (Delgado, Calvo, & Torres, ; Grisvard, Lemullois, Morin, & Baroin‐Tourancheau, ; Pomajbíková et al, ; Rios, Sarmiento, Torres, & Fedriani, ; Tadao & Fumikazu, ). During encystment, somatic ciliature and microtubular organelles dedifferentiate; tubulin gene expression may also be decreased due to the decline in demand; the cyst walls gradually form; the shape of the macronucleus is often changed, along with chromatin condensation; and autophagic activity occurs in cytoplasmic organelles, namely, lysosomes and autophagic vesicles (Aslan, Küçükoğlu, & Arslanyolu, ; Cavaleiro, Fernandes, Silva‐Neto, & Soares, ; Li et al, ; Slabodnick et al, ; Wang et al, ; Wang, Wu, Fan, & Gu, ). During excystment, the ciliature is regenerated or reactivated, often with the activation of an excystment vacuole; certain genes and proteins related to initiating the excystment process and reconstructing the vegetative cell structure are upregulated (Funadani, Suetomo, & Matsuoka, ; Grisvard et al, ; Müller, ; Sogame et al, ).…”