Haptoglobin is a plasma protein of mammals that plays a crucial role in vascular homeostasis by binding free haemoglobin released from ruptured red blood cells. Trypanosoma brucei can exploit this by internalising haptoglobin-haemoglobin complex to acquire host haem. Here, we investigated the impact of haptoglobin deficiency (Hp-/-) on T. brucei brucei infection and the parasite´s capacity to internalise haemoglobin in a Hp-/- mouse model. The infected Hp-/- mice exhibited normal disease progression, with minimal weight loss and no apparent organ pathology, similarly to control mice. While the proteomic profile of mouse sera significantly changed in response to T. b. brucei, no differences in the infection response markers of blood plasma between Hp-/- and control Black mice were observed. Similarly, very few quantitative differences were observed between the proteomes of parasites harvested from Hp-/- and Black mice, including both endogenous proteins and internalised host proteins. While haptoglobin was indeed absent from parasites isolated from Hp-/-mice, haemoglobin peptides were unexpectedly detected in parasites from both Hp-/- and Black mice. Combined, the data support the dispensability of haptoglobin for haemoglobin internalisation by T. b. brucei during infection in mice. Since the trypanosomes knock-outs for their haptoglobin-haemoglobin receptor (HpHbR) internalised significantly less haemoglobin from Hp-/- mice compared to those isolated from Black mice, it suggests that T. b. brucei employs also an HpHbR-independent haptoglobin-mediated mode for haemoglobin internalisation. Our study reveals a so-far hidden flexibility of haemoglobin acquisition by T. b. brucei and offers novel insights into alternative haemoglobin uptake pathways.

• Klíčová slova
• Trypanosoma, acute phase protein, blood markers, haemoglobin (Hb), haptoglobin (Hp), infection,
• MeSH
• haptoglobiny * genetika   metabolismus   MeSH
• hemoglobiny * metabolismus   MeSH
• modely nemocí na zvířatech MeSH
• myši inbrední C57BL MeSH
• myši knockoutované * MeSH
• myši MeSH
• proteomika metody   MeSH
• Trypanosoma brucei brucei * metabolismus   MeSH
• trypanozomóza africká * parazitologie   imunologie   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• haptoglobin-hemoglobin complex MeSH Prohlížeč
• haptoglobiny * MeSH
• hemoglobiny * MeSH

Ferredoxins comprise a large family of iron-sulfur (Fe-S) proteins that shuttle electrons in diverse biological processes. Human mitochondria contain two isoforms of [2Fe-2S] ferredoxins, FDX1 (aka adrenodoxin) and FDX2, with known functions in cytochrome P450-dependent steroid transformations and Fe-S protein biogenesis. Here, we show that only FDX2, but not FDX1, is involved in Fe-S protein maturation. Vice versa, FDX1 is specific not only for steroidogenesis, but also for heme a and lipoyl cofactor biosyntheses. In the latter pathway, FDX1 provides electrons to kickstart the radical chain reaction catalyzed by lipoyl synthase. We also identified lipoylation as a target of the toxic antitumor copper ionophore elesclomol. Finally, the striking target specificity of each ferredoxin was assigned to small conserved sequence motifs. Swapping these motifs changed the target specificity of these electron donors. Together, our findings identify new biochemical tasks of mitochondrial ferredoxins and provide structural insights into their functional specificity.

• MeSH
• ferredoxiny * MeSH
• lidé MeSH
• mitochondrie metabolismus   MeSH
• protein - isoformy metabolismus   MeSH
• proteiny obsahující železo a síru * metabolismus   MeSH
• systém (enzymů) cytochromů P-450 metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• ferredoxiny * MeSH
• protein - isoformy MeSH
• proteiny obsahující železo a síru * MeSH
• systém (enzymů) cytochromů P-450 MeSH

Euglena gracilis is a metabolically flexible, photosynthetic, and adaptable free-living protist of considerable environmental importance and biotechnological value. By label-free liquid chromatography tandem mass spectrometry, a total of 1,786 proteins were identified from the E. gracilis purified mitochondria, representing one of the largest mitochondrial proteomes so far described. Despite this apparent complexity, protein machinery responsible for the extensive RNA editing, splicing, and processing in the sister clades diplonemids and kinetoplastids is absent. This strongly suggests that the complex mechanisms of mitochondrial gene expression in diplonemids and kinetoplastids occurred late in euglenozoan evolution, arising independently. By contrast, the alternative oxidase pathway and numerous ribosomal subunits presumed to be specific for parasitic trypanosomes are present in E. gracilis. We investigated the evolution of unexplored protein families, including import complexes, cristae formation proteins, and translation termination factors, as well as canonical and unique metabolic pathways. We additionally compare this mitoproteome with the transcriptome of Eutreptiella gymnastica, illuminating conserved features of Euglenida mitochondria as well as those exclusive to E. gracilis. This is the first mitochondrial proteome of a free-living protist from the Excavata and one of few available for protists as a whole. This study alters our views of the evolution of the mitochondrion and indicates early emergence of complexity within euglenozoan mitochondria, independent of parasitism.

• Klíčová slova
• Euglena gracilis, Euglenozoa, mitochondria, proteome, protist,
• MeSH
• Euglena gracilis metabolismus   MeSH
• mitochondriální proteiny metabolismus   MeSH
• proteom * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• mitochondriální proteiny MeSH
• proteom * MeSH

In this work, we studied the biochemical properties and evolutionary histories of catalase (CAT) and ascorbate peroxidase (APX), two central enzymes of reactive oxygen species detoxification, across the highly diverse clade Eugenozoa. This clade encompasses free-living phototrophic and heterotrophic flagellates, as well as obligate parasites of insects, vertebrates, and plants. We present evidence of several independent acquisitions of CAT by horizontal gene transfers and evolutionary novelties associated with the APX presence. We posit that Euglenozoa recruit these detoxifying enzymes for specific molecular tasks, such as photosynthesis in euglenids and membrane-bound peroxidase activity in kinetoplastids and some diplonemids.

• Klíčová slova
• Euglenozoa, ascorbate peroxidase, catalase, enzymatic activity, phylogeny,
• Publikační typ
• časopisecké články MeSH

Fe-S clusters are ubiquitous cofactors of proteins involved in a variety of essential cellular processes. The biogenesis of Fe-S clusters in the cytosol and their insertion into proteins is accomplished through the cytosolic iron-sulphur protein assembly (CIA) machinery. The early- and middle-acting modules of the CIA pathway concerned with the assembly and trafficking of Fe-S clusters have been previously characterised in the parasitic protist Trypanosoma brucei. In this study, we applied proteomic and genetic approaches to gain insights into the network of protein-protein interactions of the late-acting CIA targeting complex in T. brucei. All components of the canonical CIA machinery are present in T. brucei including, as in humans, two distinct CIA2 homologues TbCIA2A and TbCIA2B. These two proteins are found interacting with TbCIA1, yet the interaction is mutually exclusive, as determined by mass spectrometry. Ablation of most of the components of the CIA targeting complex by RNAi led to impaired cell growth in vitro, with the exception of TbCIA2A in procyclic form (PCF) trypanosomes. Depletion of the CIA-targeting complex was accompanied by reduced levels of protein-bound cytosolic iron and decreased activity of an Fe-S dependent enzyme in PCF trypanosomes. We demonstrate that the C-terminal domain of TbMMS19 acts as a docking site for TbCIA2B and TbCIA1, forming a trimeric complex that also interacts with target Fe-S apo-proteins and the middle-acting CIA component TbNAR1.

• MeSH
• cytosol metabolismus   MeSH
• interakční proteinové domény a motivy MeSH
• konformace proteinů MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• proteiny obsahující železo a síru chemie   metabolismus   MeSH
• protozoální proteiny chemie   metabolismus   MeSH
• Trypanosoma brucei brucei růst a vývoj   metabolismus   MeSH
• trypanozomiáza metabolismus   parazitologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proteiny obsahující železo a síru MeSH
• protozoální proteiny MeSH

The majority of established model organisms belong to the supergroup Opisthokonta, which includes yeasts and animals. While enlightening, this focus has neglected protists, organisms that represent the bulk of eukaryotic diversity and are often regarded as primitive eukaryotes. One of these is the "supergroup" Excavata, which comprises unicellular flagellates of diverse lifestyles and contains species of medical importance, such as Trichomonas, Giardia, Naegleria, Trypanosoma and Leishmania. Excavata exhibits a continuum in mitochondrial forms, ranging from classical aerobic, cristae-bearing mitochondria to mitochondria-related organelles, such as hydrogenosomes and mitosomes, to the extreme case of a complete absence of the organelle. All forms of mitochondria house a machinery for the assembly of Fe-S clusters, ancient cofactors required in various biochemical activities needed to sustain every extant cell. In this review, we survey what is known about the Fe-S cluster assembly in the supergroup Excavata. We aim to bring attention to the diversity found in this group, reflected in gene losses and gains that have shaped the Fe-S cluster biogenesis pathways.

• Klíčová slova
• Evolution, Excavata, Fe–S cluster, Mitochondria,
• MeSH
• Eukaryota cytologie   metabolismus   MeSH
• mitochondrie metabolismus   MeSH
• proteiny obsahující železo a síru metabolismus   MeSH
• železo metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• proteiny obsahující železo a síru MeSH
• železo MeSH

Dynamins and dynamin-like proteins (DLPs) belong to a family of large GTPases involved in membrane remodelling events. These include both fusion and fission processes with different dynamin proteins often having a specialised function within the same organism. Trypanosoma brucei is thought to have only one multifunctional DLP (TbDLP). While this was initially reported to function in mitochondrial division only, an additional role in endocytosis and cytokinesis was later also proposed. Since there are two copies of TbDLP present in the trypanosome genome, we investigated potential functional differences between these two paralogs by re-expressing either protein in a TbDLP RNAi background. These paralogs, called TbDLP1 and TbDLP2, are almost identical bar a few amino acid substitutions. Our results, based on cell lines carrying tagged and RNAi-resistant versions of each protein, show that overexpression of TbDLP1 alone is able to rescue the observed endocytosis and growth defects in the mammalian bloodstream form (BSF) of the parasite. While TbDLP2 shows no rescue in our experiments in BSF, this might also be due to lower expression levels of the protein in this life stage. In contrast, both TbDLP proteins apparently play more complementary roles in the insect procyclic form (PCF) since neither TbDLP1 nor TbDLP2 alone can fully restore wildtype growth and morphology in TbDLP-depleted parasites.

• MeSH
• buněčné linie MeSH
• dynaminy chemie   genetika   metabolismus   MeSH
• protozoální proteiny chemie   genetika   metabolismus   MeSH
• RNA interference MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie aminokyselin MeSH
• Trypanosoma brucei brucei metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• dynaminy MeSH
• protozoální proteiny MeSH

The human parasite Trypanosoma brucei does not synthesize heme de novo and instead relies entirely on heme supplied by its vertebrate host or its insect vector, the tsetse fly. In the host bloodstream T. brucei scavenges heme via haptoglobin-hemoglobin (HpHb) receptor-mediated endocytosis occurring in the flagellar pocket. However, in the procyclic developmental stage, in which T. brucei is confined to the tsetse fly midgut, this receptor is apparently not expressed, suggesting that T. brucei takes up heme by a different, unknown route. To define this alternative route, we functionally characterized heme transporter TbHrg in the procyclic stage. RNAi-induced down-regulation of TbHrg in heme-limited culture conditions resulted in slower proliferation, decreased cellular heme, and marked changes in cellular morphology so that the cells resemble mesocyclic trypomastigotes. Nevertheless, the TbHrg KO developed normally in the tsetse flies at rates comparable with wild-type cells. T. brucei cells overexpressing TbHrg displayed up-regulation of the early procyclin GPEET and down-regulation of the late procyclin EP1, two proteins coating the T. brucei surface in the procyclic stage. Light microscopy of immunostained TbHrg indicated localization to the flagellar membrane, and scanning electron microscopy revealed more intense TbHrg accumulation toward the flagellar pocket. Based on these findings, we postulate that T. brucei senses heme levels via the flagellar TbHrg protein. Heme deprivation in the tsetse fly anterior midgut might represent an environmental stimulus involved in the transformation of this important human parasite, possibly through metabolic remodeling.

• Klíčová slova
• differentiation, flagellum, heme, import, parasite, procyclin, transporter, trypanosome,
• MeSH
• biologický transport MeSH
• down regulace MeSH
• flagella metabolismus   MeSH
• hem metabolismus   MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• membránové transportní proteiny metabolismus   MeSH
• mikroskopie elektronová rastrovací MeSH
• moucha tse-tse parazitologie   MeSH
• proliferace buněk MeSH
• protozoální proteiny metabolismus   MeSH
• receptory buněčného povrchu metabolismus   MeSH
• RNA interference MeSH
• sekvence aminokyselin MeSH
• stadia vývoje MeSH
• transgeny MeSH
• Trypanosoma brucei brucei metabolismus   MeSH
• vývojová regulace genové exprese MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• hem MeSH
• hemoglobin-haptoglobin receptor MeSH Prohlížeč
• membránové transportní proteiny MeSH
• protozoální proteiny MeSH
• receptory buněčného povrchu MeSH

The cell shape of Trypanosoma brucei is influenced by flagellum-to-cell-body attachment through a specialised structure - the flagellum attachment zone (FAZ). T. brucei exhibits numerous morphological forms during its life cycle and, at each stage, the FAZ length varies. We have analysed FLAM3, a large protein that localises to the FAZ region within the old and new flagellum. Ablation of FLAM3 expression causes a reduction in FAZ length; however, this has remarkably different consequences in the tsetse procyclic form versus the mammalian bloodstream form. In procyclic form cells FLAM3 RNAi results in the transition to an epimastigote-like shape, whereas in bloodstream form cells a severe cytokinesis defect associated with flagellum detachment is observed. Moreover, we demonstrate that the amount of FLAM3 and its localisation is dependent on ClpGM6 expression and vice versa. This evidence demonstrates that FAZ is a key regulator of trypanosome shape, with experimental perturbations being life cycle form dependent. An evolutionary cell biology explanation suggests that these differences are a reflection of the division process, the cytoskeleton and intrinsic structural plasticity of particular life cycle forms.

• Klíčová slova
• Flagellum attachment zone, Morphogenesis, Trypanosomes,
• MeSH
• cilie genetika   metabolismus   MeSH
• cytokineze genetika   MeSH
• cytoskelet genetika   metabolismus   MeSH
• flagella genetika   metabolismus   MeSH
• mikrotubuly genetika   MeSH
• protozoální proteiny genetika   metabolismus   MeSH
• stadia vývoje genetika   MeSH
• Trypanosoma brucei brucei genetika   růst a vývoj   MeSH
• tvar buňky genetika   MeSH
• vývojová regulace genové exprese MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• protozoální proteiny MeSH

Trypanosoma brucei has a complex life cycle during which its single mitochondrion is subjected to major metabolic and morphological changes. While the procyclic stage (PS) of the insect vector contains a large and reticulated mitochondrion, its counterpart in the bloodstream stage (BS) parasitizing mammals is highly reduced and seems to be devoid of most functions. We show here that key Fe-S cluster assembly proteins are still present and active in this organelle and that produced clusters are incorporated into overexpressed enzymes. Importantly, the cysteine desulfurase Nfs, equipped with the nuclear localization signal, was detected in the nucleolus of both T. brucei life stages. The scaffold protein Isu, an interacting partner of Nfs, was also found to have a dual localization in the mitochondrion and the nucleolus, while frataxin and both ferredoxins are confined to the mitochondrion. Moreover, upon depletion of Isu, cytosolic tRNA thiolation dropped in the PS but not BS parasites.

• MeSH
• aktivní transport - buněčné jádro MeSH
• buněčné jádro metabolismus   MeSH
• ferredoxiny metabolismus   MeSH
• frataxin MeSH
• jaderné lokalizační signály MeSH
• lyasy štěpící vazby C-S chemie   genetika   metabolismus   MeSH
• mitochondriální proteiny metabolismus   MeSH
• mitochondrie metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• multimerizace proteinu MeSH
• proteiny asociované s jadernou matrix chemie   genetika   metabolismus   MeSH
• proteiny vázající železo metabolismus   MeSH
• protozoální proteiny chemie   genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• Trypanosoma brucei brucei enzymologie   genetika   metabolismus   MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cysteine desulfurase MeSH Prohlížeč
• ferredoxiny MeSH
• jaderné lokalizační signály MeSH
• lyasy štěpící vazby C-S MeSH
• mitochondriální proteiny MeSH
• proteiny asociované s jadernou matrix MeSH
• proteiny vázající železo MeSH
• protozoální proteiny MeSH