Branched late-steps of the cytosolic iron-sulphur cluster assembly machinery of Trypanosoma brucei

Support from the Czech Grant Agency (16-18699S to JL) and partial funding by CAPES/Science without Borders (BEX1333/13-5 to MLT) is kindly acknowledged. We are grateful to the University of St. Andrews mass spectrometry facility for collecting and processing MS data and to other members of the TKS and SM groups for their assistance with this project. Work of RL and AJP was supported by the Deutsche Forschungsgemeinschaft (Koselleck grant (to RL) and SPP 1927) and a Coordenação de aperfeiçoamento de pessoal de nivel superior (CAPES - 1333/2013-05) for the financial support to this project. We acknowledge networking support from the COST Action FeSBioNet (Contract CA15133). ERD Funds, The Czech Ministry of Education, project OPVVV 16_019/0000759 to JL.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.Publisher PDFPeer reviewe

A leucine aminopeptidase is involved in kinetoplast DNA segregation in <i>Trypanosoma brucei</i>

The kinetoplast (k), the uniquely packaged mitochondrial DNA of trypanosomatid protists is formed by a catenated network of minicircles and maxicircles that divide and segregate once each cell cycle. Although many proteins involved in kDNA replication and segregation are now known, several key steps in the replication mechanism remain uncharacterized at the molecular level, one of which is the nabelschnur or umbilicus, a prominent structure which in the mammalian parasite Trypanosoma brucei connects the daughter kDNA networks prior to their segregation. Here we characterize an M17 family leucyl aminopeptidase metalloprotease, termed TbLAP1, which specifically localizes to the kDNA disk and the nabelschur and represents the first described protein found in this structure. We show that TbLAP1 is required for correct segregation of kDNA, with knockdown resulting in delayed cytokinesis and ectopic expression leading to kDNA loss and decreased cell proliferation. We propose that TbLAP1 is required for efficient kDNA division and specifically participates in the separation of daughter kDNA networks

Impact of COVID-19 on cardiovascular testing in the United States versus the rest of the world

Objectives: This study sought to quantify and compare the decline in volumes of cardiovascular procedures between the United States and non-US institutions during the early phase of the coronavirus disease-2019 (COVID-19) pandemic. Background: The COVID-19 pandemic has disrupted the care of many non-COVID-19 illnesses. Reductions in diagnostic cardiovascular testing around the world have led to concerns over the implications of reduced testing for cardiovascular disease (CVD) morbidity and mortality. Methods: Data were submitted to the INCAPS-COVID (International Atomic Energy Agency Non-Invasive Cardiology Protocols Study of COVID-19), a multinational registry comprising 909 institutions in 108 countries (including 155 facilities in 40 U.S. states), assessing the impact of the COVID-19 pandemic on volumes of diagnostic cardiovascular procedures. Data were obtained for April 2020 and compared with volumes of baseline procedures from March 2019. We compared laboratory characteristics, practices, and procedure volumes between U.S. and non-U.S. facilities and between U.S. geographic regions and identified factors associated with volume reduction in the United States. Results: Reductions in the volumes of procedures in the United States were similar to those in non-U.S. facilities (68% vs. 63%, respectively; p = 0.237), although U.S. facilities reported greater reductions in invasive coronary angiography (69% vs. 53%, respectively; p < 0.001). Significantly more U.S. facilities reported increased use of telehealth and patient screening measures than non-U.S. facilities, such as temperature checks, symptom screenings, and COVID-19 testing. Reductions in volumes of procedures differed between U.S. regions, with larger declines observed in the Northeast (76%) and Midwest (74%) than in the South (62%) and West (44%). Prevalence of COVID-19, staff redeployments, outpatient centers, and urban centers were associated with greater reductions in volume in U.S. facilities in a multivariable analysis. Conclusions: We observed marked reductions in U.S. cardiovascular testing in the early phase of the pandemic and significant variability between U.S. regions. The association between reductions of volumes and COVID-19 prevalence in the United States highlighted the need for proactive efforts to maintain access to cardiovascular testing in areas most affected by outbreaks of COVID-19 infection

From simple to supercomplex: mitochondrial genomes of euglenozoan protists [version 2; referees: 2 approved]

Mitochondria are double membrane organelles of endosymbiotic origin, best known for constituting the centre of energetics of a eukaryotic cell. They contain their own mitochondrial genome, which as a consequence of gradual reduction during evolution typically contains less than two dozens of genes. In this review, we highlight the extremely diverse architecture of mitochondrial genomes and mechanisms of gene expression between the three sister groups constituting the phylum Euglenozoa - Euglenida, Diplonemea and Kinetoplastea. The earliest diverging euglenids possess a simplified mitochondrial genome and a conventional gene expression, whereas both are highly complex in the two other groups. The expression of their mitochondrial-encoded proteins requires extensive post-transcriptional modifications guided by complex protein machineries and multiple small RNA molecules. Moreover, the least studied diplonemids, which have been recently discovered as a highly abundant component of the world ocean plankton, possess one of the most complicated mitochondrial genome organisations known to date

The free-living flagellate Paratrimastix pyriformis uses a distinct mitochondrial carrier to balance adenine nucleotide pools

Paratrimastix pyriformis is a free-living flagellate thriving in low-oxygen freshwater sediments. It belongs to the group Metamonada along with human parasites, such as Giardia and Trichomonas. Like other metamonads, P. pyriformis has a mitochondrion-related organelle (MRO) which in this protist is primarily involved in one carbon folate metabolism. The MRO contains four members of the solute carrier family 25 (SLC25) respon sible for the exchange of metabolites across the mitochondrial inner membrane. Here, we characterise the function of the adenine nucleotide carrier PpMC1 by thermostability shift and transport assays. We show that it transports ATP, ADP and, to a lesser extent, AMP, but not phosphate. The carrier is distinct in function and origin from both ADP/ATP carriers and ATP-Mg/phosphate carriers, and it most likely represents a distinct class of adenine nucleotide carriers.publishedVersio

Functional characterization of TbMCP5, a conserved and essential ADP/ATP carrier present in the mitochondrion of the human pathogen Trypanosoma brucei

Trypanosoma brucei is a kinetoplastid parasite of medical and veterinary importance. Its digenetic life cycle alternates between the bloodstream form in the mammalian host and the procyclic form (PCF) in the bloodsucking insect vector, the tsetse fly. PCF trypanosomes rely in the glucose-depleted environment of the insect vector primarily on the mitochondrial oxidative phosphorylation of proline for their cellular ATP provision. We previously identified two T. brucei mitochondrial carrier family proteins, TbMCP5 and TbMCP15, with significant sequence similarity to functionally characterized ADP/ATP carriers from other eukaryotes. Comprehensive sequence analysis confirmed that TbMCP5 contains canonical ADP/ATP carrier sequence features, whereas they are not conserved in TbMCP15. Heterologous expression in the ANC-deficient yeast strain JL1Δ2Δ3u - revealed that only TbMCP5 was able to restore its growth on the non-fermentable carbon source lactate. Transport studies in yeast mitochondria showed that TbMCP5 has biochemical properties and ADP/ATP exchange kinetics similar to those of Anc2p, the prototypical ADP/ATP carrier of S. cerevisiae. Immunofluorescence microscopy and Western blot analysis confirmed that TbMCP5 is e xclusively mitochondrial and is differentially expressed with 4.5-fold more TbMCP5 in the procyclic form of the parasite. Silencing of TbMCP5 expression in PCF T. brucei revealed that this ADP/ATP carrier is essential for parasite growth, particularly when depending on proline for energy generation. Moreover, ADP/ATP exchange in isolated T. brucei mitochondria was eliminated upon TbMCP5 depletion. These results confirmed that TbMCP5 functions as the main ADP/ATP carrier in the trypanosome mitochondrion. The important role of TbMCP5 in the T. brucei energy metabolism is further discussed. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc

Immunofluorescence of cells ectopically expressing the TbLAP1-HA protein, double labeled with polyclonal anti-HA (green) and anti-TAC102 antibodies (red).

<p>The latter antibody visualizes the dynamics of the tripartite attachment complex (TAC). DAPI (blue) shows the location of the nucleus and kDNA; arrowheads and asterisks denote aberrant kDNA. Squares denote area displayed as zoom. B) After 2 hrs (A) and 6 hrs of ectopic expression of TbLAP1-HA (B), the apparition of 1K2Ndiv cells (not yet segregated cells with kDNA in division) became evident as depicted by two cells failing to segregate their already divided kDNAs; note the structure of the nabelschnur holding the kDNAs and TAC together at the posterior end of the cell. Division of TAC and nucleus occurred, as shown by two TAC102 signals and two nuclei, without prior segregation of the TAC structures and the kDNAs, which remained at the posterior end of the cell (C). Representative cell displaying accumulation of TbLAP1-HA around aberrantly sized kDNAs (D). Scale bars: 1 μm.</p

A model for the mutual relationships between the basal body, tripartite attachment complex and kDNA.

<p>Basal body (yellow), flagellum (black line), tripartite attachment complex (TAC) (red), kDNA and nucleus (blue), and TbLAP1 (green) are depicted during the cell cycle of procyclic <i>T</i>. <i>brucei</i>. (A) 1N1K interphase cells bearing one basal body, a flagellum and a single TAC connected with kDNA and TbLAP1. (B) Cells in the earliest stages of division, with basal and pro-basal bodies divided and started to segregate, and TAC division commenced; the growth of the new flagellum becomes evident; kDNA displays an elongated “dumbbell” shape, which reflects its replication; TbLAP1 co-localizes with kDNA. (C) Later stage where kDNA replication is complete and the progeny kDNA networks have divided and appear in a perpendicular position to one another; TbLAP1 is present in the progeny kDNAs and in the nabelschnur; basal and pro-basal bodies proceed with their segregation. (D) Later still, segregation of the newly divided kDNAs continues, together with respective TAC and basal bodies; the nabelschnur begins to fade. (E) Following nuclear DNA replication and segregation, newly divided nuclei appear, as the segregation of kDNA continues. (F) 2K2N cells just prior to cytokinesis, with nuclei completely divided with kDNA and associated structures aligned for the upcoming cell division.</p

Characterization of procyclic <i>T</i>. <i>brucei</i> expressing ectopically TbLAP1-HA.

<p>(A) Growth of trypanosomes in SDM-79 in the presence (dotted line) and absence (continuous line) of tetracycline (Tet). (B) DAPI counts in induced cells with Tet for ectopic expression of TbLAP1-HA. Two hundred cells per time point were counted and used for the analysis. (C) Western blot analysis of ectopically expressed TbLAP1-HA using monoclonal anti-tubulin antibody as a loading control. 5 x 10⁶ cells per sample were loaded per well. (D) Mitochondrial membrane potential measurement using TMRE of uninduced (black line) and induced (red line) cells expressing TbLAP1-HA (2, 6, 24, 72 and 96 hrs of induction). The measurement is a representative of an assay performed in three independent experiments.</p

Trypanosomal mitochondrial intermediate peptidase does not behave as a classical mitochondrial processing peptidase

<div><p>Upon their translocation into the mitochondrial matrix, the N-terminal pre-sequence of nuclear-encoded proteins undergoes cleavage by mitochondrial processing peptidases. Some proteins require more than a single processing step, which involves several peptidases. Down-regulation of the putative <i>Trypanosoma brucei</i> mitochondrial intermediate peptidase (MIP) homolog by RNAi renders the cells unable to grow after 48 hours of induction. Ablation of MIP results in the accumulation of the precursor of the trypanosomatid-specific trCOIV protein, the largest nuclear-encoded subunit of the cytochrome <i>c</i> oxidase complex in this flagellate. However, the trCOIV precursor of the same size accumulates also in trypanosomes in which either alpha or beta subunits of the mitochondrial processing peptidase (MPP) have been depleted. Using a chimeric protein that consists of the N-terminal sequence of a putative subunit of respiratory complex I fused to a yellow fluorescent protein, we assessed the accumulation of the precursor protein in trypanosomes, in which RNAi was induced against the alpha or beta subunits of MPP or MIP. The observed accumulation of precursors indicates MIP depletion affects the activity of the cannonical MPP, or at least one of its subunits.</p></div