Presence of a Poly(A) Binding Protein and Two Proteins with Cell Cycle-Dependent Phosphorylation in Crithidia fasciculata mRNA Cycling Sequence Binding Protein II

Crithidia fasciculata cycling sequence binding proteins (CSBP) have been shown to bind with high specificity to sequence elements present in several mRNAs that accumulate periodically during the cell cycle. The first described CSBP has subunits of 35.6 (CSBPA) and 42 kDa (CSBPB). A second distinct binding protein termed CSBP II has been purified from CSBPA null mutant cells, lacking both CSBPA and CSBPB proteins, and contains three major polypeptides with predicted molecular masses of 63, 44.5, and 33 kDa. Polypeptides of identical size were radiolabeled in UV cross-linking assays performed with purified CSBP II and (32)P-labeled RNA probes containing six copies of the cycling sequence. The CSBP II binding activity was found to cycle in parallel with target mRNA levels during progression through the cell cycle. We have cloned genes encoding these three CSBP II proteins, termed RBP63, RBP45, and RBP33, and characterized their binding properties. The RBP63 protein is a member of the poly(A) binding protein family. Homologs of RBP45 and RBP33 proteins were found only among the kinetoplastids. Both RBP45 and RBP33 proteins and their homologs have a conserved carboxy-terminal half that contains a PSP1-like domain. All three CSBP II proteins show specificity for binding the wild-type cycling sequence in vitro. RBP45 and RBP33 are phosphoproteins, and RBP45 has been found to bind in vivo specifically to target mRNA containing cycling sequences. The levels of phosphorylation of both RBP45 and RBP33 were found to cycle during the cell cycle

A novel endonuclease from kinetoplastid hemoflagellated protozoan parasite Leishmania

A nuclease activity has been purified from the nuclei-kinetoplast fraction of Leishmania. This enzyme, termed endonuclease M (Endo M), is shown by electrophoresis in a denaturing polyacrylamide gel to be associated with a single polypeptide of molecular mass 52 kDa. Physical analysis of the enzyme indicates that it has a sedimentation coefficient S20,w of 4.5S, a Stoke's radius of 32.5 A, and a native molecular mass of 53 kDa. The final Mono Q purified Endo M possesses both DNase and RNase activities. It acts as an endonuclease by introducing random single-stranded nicks into the supercolled DNA molecules, that often leads to its linearization due to nicking at the opposite strands, and subsequent degradation of the DNA with further incubation. Single-stranded DNA is twice preferred to double-stranded DNA as substrate. Single-stranded RNA is also degraded rapidly and is competitive as a substrate with single-stranded DNA. RNA: DNA hybrids, however, are largely resistant to the Endo M digestion

Telomere, telomerase, tumorigenesis and therapy: An overview

839-842The ends of chromosome in higher eukaryote are termed telomere. The DNAs present at that part of chromosome is called telomeric DNA. Telomeric DNA consists of tandemly repeated DNA sequences. The replication of the ends of chromosomes is not controlled by conventional DNA polymerases rather a special kind of enzyme is involved in this process. It is a ribonucleoprotein and known as telomerase. Cells in senescence stage face telomeric crisis that leads to loss of telomeric ends. Surveillance turns to procancer cells with increased telomerase activity which is a later consequence. Based on these facts a key di agnostic approach has been developed for detection of tumour. A novel therapy for tumour repression has been developed using telomerase inhibitors.However, these inhibitors are very much effective for solid tumour therapy and conceptually will not work on hematological malignancies

Diospyrin, A Bisnaphthoquinone: A Novel Inhibitor of Type I DNA Topoisomerase of Leishmania donovani

Diospyrin is a plant product that has significant inhibitory effect on the growth of Leishmania donovani promastigotes. This compound inhibits the catalytic activity of DNA topoisomerase I of the parasite. Like camptothecin, it induces topoisomerase I mediated DNA cleavage in vitro. Treatment of DNA with diospyrin before addition of topoisomerase I has no effect. Preincubation of topoisomerase I with diospyrin before the addition of DNA in the relaxation reaction increases this inhibition. Our results suggest that this bis-naphthoquinone compound exerts its inhibitory effect by binding with the enzyme and stabilizing the topoisomerase I-DNA “cleavable complex.” Diospyrin is a specific inhibitor of the parasitic topoisomerase I. It does not inhibit type II topoisomerase of L. donovani and requires much higher concentrations to inhibit type I topoisomerase of calf thymus. The potent inhibitory effect of diospyrin on type I DNAtopoisomerase from L. donovani can be exploited for rational drug design in human leishmaniasis

LFR1 Ferric Iron Reductase of Leishmania amazonensis Is Essential for the Generation of Infective Parasite Forms

The protozoan parasite Leishmania is the causative agent of serious human infections worldwide. the parasites alternate between insect and vertebrate hosts and cause disease by invading macrophages, where they replicate. Parasites lacking the ferrous iron transporter LIT1 cannot grow intracellularly, indicating that a plasma membrane-associated mechanism for iron uptake is essential for the establishment of infections. Here, we identify and functionally characterize a second member of the Leishmania iron acquisition pathway, the ferric iron reductase LFR1. the LFR1 gene is up-regulated under iron deprivation and accounts for all the detectable ferric reductase activity exposed on the surface of Leishmania amazonensis. LFR1 null mutants grow normally as promastigote insect stages but are defective in differentiation into the vertebrate infective forms, metacyclic promastigotes and amastigotes. LFR1 overexpression partially restores the abnormal morphology of infective stages but markedly reduces parasite viability, precluding its ability to rescue LFR1 null replication in macrophages. However, LFR1 overexpression is not toxic for amastigotes lacking the ferrous iron transporter LIT1 and rescues their growth defect. in addition, the intracellular growth of both LFR1 and LIT1 null parasites is rescued in macrophages loaded with exogenous iron. This indicates that the Fe(3+) reductase LFR1 functions upstream of LIT1 and suggests that LFR1 overexpression results in excessive Fe(2+) production, which impairs parasite viability after intracellular transport by LIT1.National Institutes of HealthUniv Maryland, Dept Cell Biol & Mol Genet, College Pk, MD 20742 USAUniversidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, BR-04023062 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, BR-04023062 São Paulo, BrazilNational Institutes of Health: R01 AI067979Web of Scienc

The iron-dependent mitochondrial superoxide dismutase SODA promotesLeishmania virulence

Menezes, Juliana Perrone Bezerra de. “Documento produzido em parceria ou por autor vinculado à Fiocruz, mas não consta à informação no documento”.Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2018-03-26T14:14:29Z No. of bitstreams: 1 Mittra B The iron-dependent mitochondrial superoxide....pdf: 3577331 bytes, checksum: 4212fdc48af97da40a383cd14d24ead8 (MD5)Approved for entry into archive by Ana Maria Fiscina Sampaio ([email protected]) on 2018-03-26T14:34:09Z (GMT) No. of bitstreams: 1 Mittra B The iron-dependent mitochondrial superoxide....pdf: 3577331 bytes, checksum: 4212fdc48af97da40a383cd14d24ead8 (MD5)Made available in DSpace on 2018-03-26T14:34:09Z (GMT). No. of bitstreams: 1 Mittra B The iron-dependent mitochondrial superoxide....pdf: 3577331 bytes, checksum: 4212fdc48af97da40a383cd14d24ead8 (MD5) Previous issue date: 2017National Institutes of Health Grant R01 AI067979 (to N. W. A.).University of Maryland. Department of Cell Biology and Molecular Genetics. Maryland, USAUniversity of Maryland. Department of Cell Biology and Molecular Genetics. Maryland, USAUniversity of Maryland. Department of Cell Biology and Molecular Genetics. Maryland, USAUniversity of Maryland. Department of Cell Biology and Molecular Genetics. Maryland, USAUniversity of Maryland. Department of Cell Biology and Molecular Genetics. Maryland, USALeishmaniasis is one of the leading globally neglected diseases, affecting millions of people worldwide.Leishmaniainfection depends on the ability of insect-transmitted metacyclic promastigotes to invade mammalian hosts, differentiate into amastigotes, and replicate inside macrophages. To counter the hostile oxidative environment inside macrophages, these protozoans contain anti-oxidant systems that include iron-dependent superoxide dismutases (SODs) in mitochondria and glycosomes. Increasing evidence suggests that in addition to this protective role,Leishmaniamitochondrial SOD may also initiate H2O2-mediated redox signaling that regulates gene expression and metabolic changes associated with differentiation into virulent forms. To investigate this hypothesis, we examined the specific role of SODA, the mitochondrial SOD isoform inLeishmania amazonensisOur inability to generateL. amazonensis SODAnull mutants and the lethal phenotype observed following RNAi-mediated silencing of theTrypanosoma brucei SODAortholog suggests that SODA is essential for trypanosomatid survival.L. amazonensismetacyclic promastigotes lacking oneSODAallele failed to replicate in macrophages and were severely attenuated in their ability to generate cutaneous lesions in mice. Reduced expression of SODA also resulted in mitochondrial oxidative damage and failure ofSODA/ΔsodApromastigotes to differentiate into axenic amastigotes. SODA expression above a critical threshold was also required for the development of metacyclic promastigotes, asSODA/ΔsodAcultures were strongly depleted in this infective form and more susceptible to reactive oxygen species (ROS)-induced stress. Collectively, our data suggest that SODA promotesLeishmaniavirulence by protecting the parasites against mitochondrion-generated oxidative stress and by initiating ROS-mediated signaling mechanisms required for the differentiation of infective forms

Trypanosoma brucei Spliced Leader RNA Maturation by the Cap 1 2′-O-Ribose Methyltransferase and SLA1 H/ACA snoRNA Pseudouridine Synthase Complex ▿ ‡

Kinetoplastid flagellates attach a 39-nucleotide spliced leader (SL) upstream of protein-coding regions in polycistronic RNA precursors through trans splicing. SL modifications include cap 2′-O-ribose methylation of the first four nucleotides and pseudouridine (ψ) formation at uracil 28. In Trypanosoma brucei, TbMTr1 performs 2′-O-ribose methylation of the first transcribed nucleotide, or cap 1. We report the characterization of an SL RNA processing complex with TbMTr1 and the SLA1 H/ACA small nucleolar ribonucleoprotein (snoRNP) particle that guides SL ψ28 formation. TbMTr1 is in a high-molecular-weight complex containing the four conserved core proteins of H/ACA snoRNPs, a kinetoplastid-specific protein designated methyltransferase-associated protein (TbMTAP), and the SLA1 snoRNA. TbMTAP-null lines are viable but have decreased SL RNA processing efficiency in cap methylation, 3′-end maturation, and ψ28 formation. TbMTAP is required for association between TbMTr1 and the SLA1 snoRNP but does not affect U1 small nuclear RNA methylation. A complex methylation profile in the mRNA population of TbMTAP-null lines indicates an additional effect on cap 4 methylations. The TbMTr1 complex specializes the SLA1 H/ACA snoRNP for efficient processing of multiple modifications on the SL RNA substrate

Complete Cap 4 Formation Is Not Required for Viability in Trypanosoma brucei

In kinetoplastids spliced leader (SL) RNA is trans-spliced onto the 5′ ends of all nuclear mRNAs, providing a universal exon with a unique cap. Mature SL contains an m(7)G cap, ribose 2′-O methylations on the first four nucleotides, and base methylations on nucleotides 1 and 4 (AACU). This structure is referred to as cap 4. Mutagenized SL RNAs that exhibit reduced cap 4 are trans-spliced, but these mRNAs do not associate with polysomes, suggesting a direct role in translation for cap 4, the primary SL sequence, or both. To separate SL RNA sequence alterations from cap 4 maturation, we have examined two ribose 2′-O-methyltransferases in Trypanosoma brucei. Both enzymes fall into the Rossmann fold class of methyltransferases and model into a conserved structure based on vaccinia virus homolog VP39. Knockdown of the methyltransferases individually or in combination did not affect growth rates and suggests a temporal placement in the cap 4 formation cascade: TbMT417 modifies A(2) and is not required for subsequent steps; TbMT511 methylates C(3), without which U(4) methylations are reduced. Incomplete cap 4 maturation was reflected in substrate SL and mRNA populations. Recombinant methyltransferases bind to a methyl donor and show preference for m(7)G-capped RNAs in vitro. Both enzymes reside in the nucleoplasm. Based on the cap phenotype of substrate SL stranded in the cytosol, A(2), C(3), and U(4) methylations are added after nuclear reimport of Sm protein-complexed substrate SL RNA. As mature cap 4 is dispensable for translation, cap 1 modifications and/or SL sequences are implicated in ribosomal interaction