Subrtova, K. | bioRxiv Science

bioRxiv2025

Proteolytic Processing of the Unique F1-ATPase Subunit α in Procyclic Trypanosoma brucei

The catalytic F1 domain of the mitochondrial ATP synthase is conserved across eukaryotes, with the only known exceptions found within euglenozoans. One distinctive trait identified in Trypanosoma brucei is the proteolysis of subunit that excises an internal octapeptide, resulting in functional N- and C-terminal polypeptides. The significance of this cleavage remained unclear. Here, we determined that the yeast subunit expressed in T. brucei was not proteolytically processed, despite significant structural similarities. The proteolytic recognition sequence was identified to be largely contained within the octapeptide and replacing it with a flexible linker rendered the protein resistant to cleavage. This uncleaved subunit restored growth after RNAi depletion of endogenous subunit by incorporating into F1Fo-ATP synthase complexes capable of both ATP synthesis and hydrolysis. A FRET-based assay revealed that peptides consisting of either the first or second octapeptide cleavage site experienced significantly more proteolysis when incubated with cytosolic lysates compared to mitochondrial extracts. Furthermore, expressing just the mature C-terminal polypeptide resulted in mitochondrial localization, suggesting it contains an internal targeting signal. Together, these results indicate that proteolysis of subunit occurs in the cytosol prior to mitochondrial import, highlighting a unique processing step in T. brucei ATP synthase assembly.

cell biology↗

bioRxiv2022

Repurposing of mitochondria-targeted tamoxifen: Novel anti-cancer drug exhibits potent activity against major protozoan and fungal pathogens.

Many of the currently available anti-parasitic and anti-fungal frontline drugs have severe limitations, including adverse side effects, complex administration, and increasing occurrence of resistance. The discovery and development of new therapeutic agents is a costly and lengthy process. Therefore, repurposing drugs with already established clinical application offers an attractive, fast-track approach for novel treatment options. In this study, we show that the anti-cancer drug MitoTam, a mitochondria-targeted analog of tamoxifen, efficiently eliminates a wide range of evolutionarily distinct pathogens in vitro, including pathogenic fungi, Plasmodium falciparum, and several species of trypanosomatid parasites, causative agents of debilitating neglected tropical diseases. MitoTam treatment was also effective in vivo and significantly reduced parasitemia of two medically important parasites, Leishmania mexicana and Trypanosoma brucei, in their respective animal infection models. Functional analysis in the bloodstream form of T. brucei showed that MitoTam rapidly altered mitochondrial functions, particularly affecting cellular respiration, lowering ATP levels, and dissipating mitochondrial membrane potential. Our data suggest that the mode of action of MitoTam involves disruption of the inner mitochondrial membrane, leading to rapid organelle depolarization and cell death. Altogether, MitoTam is an excellent candidate drug against several important pathogens, for which there are no efficient therapies and for which drug development is not a priority. Author SummaryMitoTam, a mitochondrially targeted analog of tamoxifen, is a promising anti-cancer candidate drug acting by accumulating in and destabilizing cell mitochondria. In this study, we analyze its effect on a wide range of evolutionarily distinct and medically important pathogens. These include a) pathogenic fungi, Candida albicans, and Cryptococcus neoformans, ubiquitous opportunistic pathogens that cause life-threatening diseases in immunocompromised or immunologically deficient individuals; b) Plasmodium falciparum, the causative agent of human malaria; and c) several species of trypanosomatid parasites such as Trypanosoma cruzi, responsible for deadly Chagas disease in South America, Trypanosoma brucei, the causative agent of sleeping sickness in Africa, and Leishmania, the etiological agent of leishmaniasis, a spectrum of diseases ranging from usually self-healing but potentially disfiguring cutaneous and mucocutaneous leishmaniasis, to visceral leishmaniasis, which is invariably fatal if left untreated. We show that MitoTam efficiently kills these parasites in laboratory conditions and in the case of trypanosomes and leishmaniases suppress or at least slow down the infection in mouse model.

microbiology↗