Biology subjects

Skodova-Sverakova, I.

Publications and source records attributed to Skodova-Sverakova, I..

6 recordsLinked to original sources

bioRxiv2026

Alternative oxidase in trypanosomatids

Background. Alternative oxidase (AOX) is a mitochondrial terminal oxidase that provides an alternative route for electron transfer, contributing to respiratory flexibility and redox homeostasis under stress conditions. In trypanosomatids, AOX displays a highly uneven distribution associated with extensive diversification of mitochondrial electron transport chains (ETCs). In addition to canonical AOX, many trypanosomatids encode an enigmatic AOX-like protein (AOX-L), whose evolutionary origin and functional significance remain unresolved. Results. Here, we investigated the evolutionary distribution, phylogenetic relationships, structural conservation, and functional divergence of AOX and AOX-L across euglenozoans. Comparative genomic analyses revealed a patchy distribution of both proteins among kinetoplastids, with multiple independent losses during evolution. Phylogenetic analyses demonstrated that AOX and AOX-L represent distinct evolutionary lineages, indicating independent origins rather than duplication-derived divergence. While AOX showed a conserved eukaryotic origin, AOX-L occupied a separate phylogenetic position and lacked key residues required for ubiquinol oxidation. Structural modelling revealed that AOX-L retained the characteristic AOX fold, predicted membrane association, and dimeric organization, but lacked the catalytic architecture necessary for enzymatic activity. Transcriptomic and biochemical analyses of four AOX-containing trypanosomatid species showed that AOX expression and activity correlated with mitochondrial ETC organization. Species lacking cytochrome-dependent complexes III and IV displayed substantially higher AOX expression and enzymatic activity, consistent with AOX functioning as the primary terminal oxidase in these lineages. Conversely, species retaining a canonical ETC exhibited lower AOX activity, suggesting a role in metabolic flexibility and redox regulation. Conclusions. Our study provides a comprehensive evolutionary and functional framework for alternative oxidases in trypanosomatids. We demonstrate that AOX and AOX-L are evolutionarily distinct proteins with different predicted functions: AOX maintains respiratory electron flow according to lineage-specific mitochondrial requirements, whereas AOX-L represents a structurally conserved but catalytically inactive protein family that may have acquired an alternative regulatory role. These findings highlight how mitochondrial respiratory components diversify during eukaryotic evolution and provide a basis for future investigations into the biological function of AOX-L.

biochemistry↗

bioRxiv2026

Interconnections with and within the trypanosomal respiratory chain revealed by complexome profiling

Proteins are frequently integrated into multicomponent complexes that execute the elaborate processes supporting life. Thus, a protein's function can only be defined by the company it keeps within a complex. Proteomes provide informative protein inventories but lack information about protein quaternary structures. Complexome profiling (CP) has been transformative in capturing the comprehensive population structure of complexes at a given moment within a cell. We have employed CP to chart the multiprotein complex landscape of two life cycle stages of Trypanosoma brucei. These data have allowed the observation of previously hidden interactions with and within the mitochondrial respiratory chain. We have found (1) an exceptional case of a SLC25 solute transporter that interacts with NADH dehydrogenase, (2) two ATP synthase subunit g paralogs that are intriguingly excluded from the enzyme's dimers, and (3) refined the known composition of ubiquinol:cytochrome c oxidoreductase by addition of missing subunits and removing an incorrectly assigned subunit, which more likely acts to insert the iron-sulfur co-factor into the complex. Further investigation into ubiquinol:cytochrome c oxidoreductase assembly revealed crosstalk between incorporation of its nuclear subunits with mitochondrial translation, possibly facilitating a hitherto unknown quality control mechanism. These discoveries demonstrate the power of our CP data for generation and testing of hypotheses about the mitochondrial and other organellar multiprotein complexes of T. brucei, a protist of medical and evolutionary importance.

cell biology↗

bioRxiv2026

A p32 family RNA editing factor acts in mitochondrial ribosome biogenesis

Biogenesis of mitochondrial ribosomes (mitoribosomes) in the unicellular parasite Trypanosoma brucei requires an exceptionally large toolkit of assembly factors, identified in stable precursors of large and small mitoribosomal subunits (mtLSU and mtSSU) by cryoEM. Here, using genetic modifications and proteomic characterization of the immunoprecipitated assemblosome, the earliest characterized mtSSU precursor, we determined that a cap of its distinctive protrusion of hitherto unknown composition consists of a p22 homotrimer. This protein was previously implicated in the uridine-insertion editing of the cytochrome c oxidase subunit II transcript. Our functional analysis confirmed this role but revealed that its ablation also causes a loss of mtSSU and a systemic reduction in mitochondrial translation, phenocopying the depletion of established mitoribosomal assembly factors. Consequently, the oxidative phosphorylation system and mitochondrial function are compromised. The p22 protein belongs to the p32 family. We showed that five of its six trypanosomal members are involved in mtSSU biogenesis. Notably, p32 proteins are associated with mitoribosomes in two other distant eukaryotic lineages. A eukaryote-wide mapping of p32 proteins documented that their presence correlates with the retention of mitochondrial genomes. Together, our findings redefine trypanosomal p22 as a dual-function coordinator of mitochondrial gene expression and reveal that the ancestral role of the p32 family is associated with mitochondrial translation.

molecular biology↗

bioRxiv2026

Distinct roles of three trypanosomal Oxa1 insertases in biogenesis of mitochondrial membrane complexes

The insertase Oxa1 is required for protein insertion into the inner mitochondrial membrane and for the biogenesis of oxidative phosphorylation complexes. While most eukaryotes encode one or two Oxa1 proteins, we identified three paralogs in Trypanosoma brucei: TbOxa1-1, TbOxa1-2, and TbOxa1-3. Knock-out of individual paralogs followed by phenotypic analyses and proteomic characterization of submitochondrial fractions revealed distinct functions. Respiratory chain complexes I and IV are primarily affected by loss of TbOxa1-1, whereas complex III and ATP synthase depend on TbOxa1-2; the ablation of TbOxa1-3 results in minor phenotypes in culture. In TbOxa1-2-depleted cells, ATP synthase biogenesis is compromised by the defective import or processing of the nuclear-encoded subunit-c, which also requires a rhomboid peptidase-like protein. Further, the ablation of TbOxa1-2 triggers accumulation of membrane proteins in the matrix, supporting its role in conservative sorting. Together, our results demonstrate that the trypanosomal Oxa1 machinery evolved a paralog-specific division of labor to manage a highly divergent mitochondrial membrane proteome.

biochemistry↗

bioRxiv2024

Characterization of novel and essential kinetoplast-associated proteins in Trypanosoma brucei

The kinetoplast is one of the defining features of kinetoplastid protists and represents a unique concentration of mitochondrial DNA. This subcellular structure is a highly complex assembly of thousands of mutually catenated, circular DNA molecules as well as up to one hundred dedicated proteins. These components work in tandem to replicate and segregate the mitochondrial genome during cellular division, additionally coordinating with the basal body and flagellum through the tripartite attachment complex (TAC) superstructure. Here, we screened the MitoTag localization repository and identified a number of previously undescribed hypothetical proteins exhibiting putative signals within the kinetoplast of Trypanosoma brucei. Through endogenous tagging we verify their association with the kinetoplast or TAC. The essentiality for several of these kinetoplast proteins (KP) was assessed by RNAi knock-downs, revealing that the newly characterized KP56, KP84 and KP86 are indispensable for growth of the procyclic stage. Additionally, KP37, KP56, and KP84 displayed alterations in the abundance of maxicircles or minicircles, while the depletion of KP84 and KP86 resulted in cell cycle alternations. Pulldown assays using the endogenously V5-tagged cell lines identified novel interactors, which were additionally subjected to endogenous tagging for subcellular localization, revealing two additional proteins (KP45 and KP66) with dual localization to the kinetoplast and throughout the mitochondrial lumen. This work represents the most extensive identification of novel KPs to date and provides a methodological pipeline for the characterization of remaining KPs to further understand this intricate molecular structure.

cell biology↗

bioRxiv2021

Anaerobic derivates of mitochondria and peroxisomes in the free-living amoeba Pelomyxa schiedti revealed by single-cell genomics

Pelomyxa schiedti is a free-living amoeba belonging to the group Archamoebae, which encompasses anaerobes bearing mitochondrion-related organelles (MROs) - hydrogenosomes in free-living Mastigamoeba balamuthi and mitosomes in the human pathogen Entamoeba histolytica. Anaerobic peroxisomes, another adaptation to anaerobic lifestyle, were identified only recently in M. balamuthi. We found evidence for both these organelles in the single-cell-derived genome and transcriptome of P. schiedti, and corresponding vesicles were tentatively revealed in electron micrographs. In silico reconstructed MRO metabolism seems similar to that of M. balamuthi harboring respiratory complex II, electron-transferring flavoprotein, partial TCA cycle running presumably in reductive direction, pyruvate:ferredoxin oxidoreductase, [FeFe]-hydrogenases, glycine cleavage system, and sulfate activation pathway. The cell disposes with an expanded set of NIF enzymes for iron sulfur cluster assembly, but their localization remains unclear. Quite contrary, out of 67 predicted peroxisomal enzymes, only four were reported also in M. balamuthi, namely peroxisomal processing peptidase, nudix hydrolase, inositol 2-dehydrogenase, and D-lactate dehydrogenase. Other putative functions of peroxisomes could be pyridoxal 5I-phosphate biosynthesis, amino acid and carbohydrate metabolism, and hydrolase activities. Future experimental evidence is necessary to define functions of this surprisingly enzyme-rich anaerobic peroxisome. Author summaryA major part of the microbial diversity cannot be cultured in isolation, and so it escapes from traditional ways of investigation. In this paper, we demonstrate the successful approach for generating good-quality genome and transcriptome drafts from a peculiar amoeba Pelomyxa schiedti using single-cell methods. P. schiedti is a member of Archamoebae clade harboring microaerobic protists including a free-living Mastigamoeba balamuthi and a human parasite Entamoeba histolytica. Mitochondria and peroxisomes represent two organelles that are most affected during adaptation to microoxic or anoxic environments. Mitochondria are known to transform to anaerobic mitochondria, hydrogenosomes, mitosomes, and various transition stages in between, all of which encompass different enzymatic capacity. Anaerobic peroxisomes have been first noticed in M. balamuthi, but their function remained unclear for now. Data obtained in this study were used for revealing the presence and for the detailed functional annotations of anaerobic derivates of mitochondria and peroxisomes in P. schiedti, which were corroborated by transmission electron microscopy.

evolutionary biology↗