TvAtg4.4 Autophagin Processes TvAtg8 Autophagy Proteins and Responds to Glucose in <em>Trichomonas vaginalis</em>

Microautophagy is a conserved cellular degradation process involving ATG proteins, with ATG4 proteases essential for processing ATG8 family proteins during autophagosome formation. In Trichomonas vaginalis, the role of autophagin proteases in processing autophagy markers TvAtg8a and TvAtg8b has not been fully characterized. In this study, we expressed and purified recombinant TvAtg4.4 and demonstrated its cysteine protease activity in vitro. TvAtg4.4 rapidly processed TvAtg8aGST and, to a lesser extent, TvAtg8bGST. Enzymatic assays confirmed substrate specificity and inhibition by cysteine protease inhibitors. TvAtg4.4 mRNA expression increased under glucose restriction, and immunolocalization showed its presence in autophagic vesicles, cytoplasm, endoplasmic reticulum, Golgi, lysosomes, hydrogenosomes, and nucleus. Colocalization with TvAtg8a and TvAtg8b supports its functional role in autophagy. The localization of TvAtg4.4 in autophagosomes and ER suggests its involvement in the cleavage of TvAtg8a and TvAtg8b after synthesis and in the delipidation or deconjugation of these proteins from the autophagosome outer membrane before autophagosome-lysosome fusion. These findings clarify the enzymatic function and cellular localization of TvAtg4.4, provide insight into autophagy mechanisms in T. vaginalis, and suggest potential novel roles for this protease in parasite biology.