Characterization of two different Asf1 histone chaperones with distinct cellular localizations and functions in Trypanosoma brucei

Abstract The anti-silencing function protein 1 (Asf1) is a chaperone that forms a complex with histones H3 and H4 facilitating dimer deposition and removal from chromatin. Most eukaryotes possess two different Asf1 chaperones but their specific functions are still unknown. Trypanosomes, a group of early-diverged eukaryotes, also have two, but more divergent Asf1 paralogs than Asf1 of higher eukaryotes. To unravel possible different functions, we characterized the two Asf1 proteins in Trypanosoma brucei. Asf1A is mainly localized in the cytosol but translocates to the nucleus in S phase. In contrast, Asf1B is predominantly localized in the nucleus, as described for other organisms. Cytosolic Asf1 knockdown results in accumulation of cells in early S phase of the cell cycle, whereas nuclear Asf1 knockdown arrests cells in S/G2 phase. Overexpression of cytosolic Asf1 increases the levels of histone H3 and H4 acetylation. In contrast to cytosolic Asf1, overexpression of nuclear Asf1 causes less pronounced growth defects in parasites exposed to genotoxic agents, prompting a function in chromatin remodeling in response to DNA damage. Only the cytosolic Asf1 interacts with recombinant H3/H4 dimers in vitro. These findings denote the early appearance in evolution of distinguishable functions for the two Asf1 chaperons in trypanosomes.

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In Vitro Characterization of Histone Chaperones using Analytical, Pull-Down and Chaperoning Assays

Journal of Visualized Experiments ◽

10.3791/63218 ◽

2021 ◽

Author(s):

Ruchir C. Bobde ◽

Ketul Saharan ◽

Somanath Baral ◽

Surajit Gandhi ◽

Archana Samal ◽

...

Keyword(s):

Histone Chaperones

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An In Vitro System Recapitulates Chromatin Remodeling at the PHO5 Promoter

Molecular and Cellular Biology ◽

10.1128/mcb.19.4.2817 ◽

1999 ◽

Vol 19 (4) ◽

pp. 2817-2827 ◽

Cited By ~ 11

Author(s):

Elizabeth S. Haswell ◽

Erin K. O’Shea

Keyword(s):

Chromatin Remodeling ◽

Chromatin Structure ◽

Nuclear Extract ◽

In Vitro System ◽

Chromatin Remodeling Complex ◽

Promoter Dna ◽

Pho5 Promoter

ABSTRACT The Saccharomyces cerevisiae gene PHO5 is an excellent system with which to study regulated changes in chromatin structure. The PHO5 promoter is packaged into four positioned nucleosomes under repressing conditions; upon induction, the structure of these nucleosomes is altered such that the promoter DNA becomes accessible to nucleases. We report here the development and characterization of an in vitro system in which partially purified PHO5 minichromosomes undergo promoter chromatin remodeling. Several hallmarks of thePHO5 chromatin transition in vivo were reproduced in this system. Chromatin remodeling of PHO5minichromosomes required the transcription factors Pho4 and Pho2, was localized to the promoter region of PHO5, and was independent of the chromatin-remodeling complex Swi-Snf. In vitro chromatin remodeling also required the addition of fractionated nuclear extract and hydrolyzable ATP. This in vitro system should serve as a useful tool for identifying the components required for this reaction and for elucidating the mechanism by which the PHO5promoter chromatin structure is changed.

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Isolation and characterization of polysomes from Trypanosoma brucei

Parasitology ◽

10.1017/s0031182000061928 ◽

1980 ◽

Vol 81 (3) ◽

pp. 537-552 ◽

Cited By ~ 15

Author(s):

J. S. Cordingley ◽

M. J. Turner

Keyword(s):

Protein Synthesis ◽

Trypanosoma Brucei ◽

Surface Antigen ◽

Isolation And Characterization ◽

Variant Surface Antigen ◽

26S Rrna

SUMMARYThe isolation of polysomes in bulk from bloodstream forms of Trypanosoma brucei is described. The polysomes are active in in vitro protein synthesis in the presence or absence of initiation inhibitors. Nascent variant surface antigen (VSA) has been detected on these polysomes using purified radio-iodinated antibody. EDTA-induced ribosomal sub-units and their large rRNA's are characterized. The 26S rRNA is nicked to produce 2 molecules which are both smaller than the 195 rRNA of the small sub-unit which is larger than that found in the majority of eukaryotic small sub-units.

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The Forkhead-associated Domain Protein Cep170 Interacts with Polo-like Kinase 1 and Serves as a Marker for Mature Centrioles

Molecular Biology of the Cell ◽

10.1091/mbc.e04-10-0939 ◽

2005 ◽

Vol 16 (3) ◽

pp. 1095-1107 ◽

Cited By ~ 151

Author(s):

Giulia Guarguaglini ◽

Peter I. Duncan ◽

York D. Stierhof ◽

Tim Holmström ◽

Stefan Duensing ◽

...

Keyword(s):

Cell Cycle ◽

Fha Domain ◽

Mother Centriole ◽

Bona Fide ◽

G2 Phase ◽

Domain Protein ◽

Interfering Rna

We report the characterization of Cep170, a forkhead-associated (FHA) domain protein of previously unknown function. Cep170 was identified in a yeast two-hybrid screen for interactors of Polo-like kinase 1 (Plk1). In human cells, Cep170 is constantly expressed throughout the cell cycle but phosphorylated during mitosis. It interacts with Plk1 in vivo and can be phosphorylated by Plk1 in vitro, suggesting that it is a physiological substrate of this kinase. Both overexpression and small interfering RNA (siRNA)-mediated depletion studies suggest a role for Cep170 in microtuble organization and cell morphology. Cep170 associates with centrosomes during interphase and with spindle microtubules during mitosis. As shown by immunoelectron microscopy, Cep170 associates with subdistal appendages, typical of the mature mother centriole. Thus, anti-Cep170 antibodies stain only one centriole during G1, S, and early G2, but two centrioles during late G2 phase of the cell cycle. We show that Cep170 labeling can be used to discriminate bona fide centriole overduplication from centriole amplification that results from aborted cell division.

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Trypanosoma brucei: characterization of protein kinases that are capable of autophosphorylation in vitro

Parasitology ◽

10.1017/s0031182000068256 ◽

1994 ◽

Vol 108 (2) ◽

pp. 161-166 ◽

Cited By ~ 13

Author(s):

G. Hide ◽

T. Graham ◽

N. Buchanan ◽

A. Tait ◽

K. Keith

Keyword(s):

Protein Kinase ◽

Protein Kinases ◽

Trypanosoma Brucei ◽

Mammalian Cells ◽

Molecular Size ◽

Regulatory Pathways ◽

Phosphate Donor ◽

Epidermal Growth

SUMMARYAutophosphorylation by protein kinases has been implicated as an important control mechanism in signal transduction and growth regulatory pathways in mammalian cells. We have set out to investigate whether any such autophosphorylating protein kinase activities can be found in Trypanosoma brucei. In order to do this, we have developed a system for characterizing such protein kinase activities using an in vitro assay. This assay was carried out by fractionation of trypanosome lysates using isoelectric focusing gel electrophoresis followed by incubation of the gel in γ32P-labelled nucleotide triphosphate and subsequent autoradiography. We have identified two classes of autophosphorylating protein kinase activities. In the first class all were dependent on ATP as the phosphate donor substrate and were all found to have a molecular size of 60 kDa. Differences in the activity of these protein kinases were observed between the bloodstream and procyclic life-cyle stages. Furthermore, the addition of mammalian epidermal growth factor to bloodstream stage lysates stimulated an additional activity. The second class of autophosphorylating protein kinases utilized GTP as the phosphate donor and were all found to be 90 kDa in size. Stage-specific differences were also observed in the activity of these protein kinases.

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ATP-Dependent Remodeling of the Spliceosome: Intragenic Suppressors of Release-Defective Mutants of Saccharomyces cerevisiae Prp22

Genetics ◽

10.1093/genetics/160.2.407 ◽

2002 ◽

Vol 160 (2) ◽

pp. 407-415 ◽

Cited By ~ 1

Author(s):

Eva Campodonico ◽

Beate Schwer

Keyword(s):

Atp Hydrolysis ◽

Splicing Factor ◽

Nonpermissive Temperature ◽

Mutant Proteins ◽

Growth Defects ◽

Isolation And Characterization ◽

Cold Sensitive ◽

Rna Unwinding

Abstract The essential splicing factor Prp22 is a DEAH-box helicase that catalyzes the release of mRNA from the spliceosome. ATP hydrolysis by Prp22 is necessary but not sufficient for spliceosome disassembly. Previous work showed that mutations in motif III (635SAT637) of Prp22 that uncouple ATP hydrolysis from spliceosome disassembly lead to severe cold-sensitive (cs) growth defects and to impaired RNA unwinding activity in vitro. The cs phenotype of S635A (635AAT) can be suppressed by intragenic mutations that restore RNA unwinding. We now report the isolation and characterization of new intragenic mutations that suppress the cold-sensitive growth phenotypes of the T637A motif III mutation (SAA), the H606A mutation in the DEAH-box (DEAA), and the R805A mutation in motif VI (804QAKGRAGR811). Whereas the T637A and H606A proteins are deficient in releasing mRNA from the spliceosome at nonpermissive temperature in vitro, the suppressor proteins have recovered mRNA release activity. To address the mechanisms of suppression, we tested ATPase and helicase activities of Prp22 suppressor mutant proteins and found that the ability to unwind a 25-bp RNA duplex was not restored in every case. This finding suggests that release of mRNA from the spliceosome is less demanding than unwinding of a 25-bp duplex RNA; the latter reaction presumably reflects the result of several successive cycles of ATP binding, hydrolysis, and unwinding. Increasing the reaction temperature allows H606A and T637A to effect mRNA release in vitro, but does not restore RNA unwinding by T637A.

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Characterization of Trypanosoma brucei bruceiS-adenosyl-l-methionine decarboxylase and its inhibition by Berenil, pentamidine and methylglyoxal bis(guanylhydrazone)

Biochemical Journal ◽

10.1042/bj2370685 ◽

1986 ◽

Vol 237 (3) ◽

pp. 685-689 ◽

Cited By ~ 93

Author(s):

A J Bitonti ◽

J A Dumont ◽

P P McCann

Keyword(s):

Trypanosoma Brucei ◽

Decarboxylase Activity ◽

Inhibitory Effects ◽

Trypanosoma Brucei Brucei ◽

Curative Effect ◽

Model Mouse ◽

Mammalian Enzyme

Trypanosoma brucei brucei S-adenosyl-L-methionine (AdoMet) decarboxylase was found to be relatively insensitive to activation by putrescine as compared with the mammalian enzyme, being stimulated by only 50% over a 10,000-fold range of putrescine concentrations. The enzyme was not stimulated by up to 10 mM-Mg2+. The Km for AdoMet was 30 microM, similar to that of other eukaryotic AdoMet decarboxylases. T.b. brucei AdoMet decarboxylase activity was apparently irreversibly inhibited in vitro by Berenil and reversibly by pentamidine and methylglyoxal bis(guanylhydrazone). Berenil also inhibited trypanosomal AdoMet decarboxylase by 70% within 4 h after administration to infected rats and markedly increased the concentration of putrescine in trypanosomes that were exposed to the drug in vivo. Spermidine and spermine blocked the curative effect of Berenil on model mouse T.b. brucei infections. This effect of the polyamines was probably not due to reversal of Berenil's inhibitory effects on the AdoMet decarboxylase.

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Characterization of Schizosaccharomyces pombeHus1: a PCNA-Related Protein That Associates with Rad1 and Rad9

Molecular and Cellular Biology ◽

10.1128/mcb.20.4.1254-1262.2000 ◽

2000 ◽

Vol 20 (4) ◽

pp. 1254-1262 ◽

Cited By ~ 140

Author(s):

Thomas Caspari ◽

Maria Dahlen ◽

Gunilla Kanter-Smoler ◽

Howard D. Lindsay ◽

Kay Hofmann ◽

...

Keyword(s):

Protein Complex ◽

Small Proportion ◽

S Phase ◽

Dna Integrity ◽

Related Protein ◽

Transient Interaction ◽

Two Hybrid

ABSTRACT Hus1 is one of six checkpoint Rad proteins required for allSchizosaccharomyces pombe DNA integrity checkpoints. MYC-tagged Hus1 reveals four discrete forms. The main form, Hus1-B, participates in a protein complex with Rad9 and Rad1, consistent with reports that Rad1-Hus1 immunoprecipitation is dependent on the rad9 + locus. A small proportion of Hus1-B is intrinsically phosphorylated in undamaged cells and more becomes phosphorylated after irradiation. Hus1-B phosphorylation is not increased in cells blocked in early S phase with hydroxyurea unless exposure is prolonged. The Rad1–Rad9–Hus1-B complex is readily detectable, but upon cofractionation of soluble extracts, the majority of each protein is not present in this complex. Indirect immunofluorescence demonstrates that Hus1 is nuclear and that this localization depends on Rad17. We show that Rad17 defines a distinct protein complex in soluble extracts that is separate from Rad1, Rad9, and Hus1. However, two-hybrid interaction, in vitro association and in vivo overexpression experiments suggest a transient interaction between Rad1 and Rad17.

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Synthesis of 1H-3-{4-[(3-Dimethylaminopropyl)aminomethyl]phenyl}-2-phenylindole and Evaluation of Its Antiprotozoal Activity

Molbank ◽

10.3390/m1060 ◽

2019 ◽

Vol 2019 (2) ◽

pp. M1060 ◽

Cited By ~ 2

Author(s):

Jean Guillon ◽

Clotilde Boudot ◽

Anita Cohen ◽

Solène Savrimoutou ◽

Sandra Rubio ◽

...

Keyword(s):

Trypanosoma Brucei ◽

Leishmania Donovani ◽

Structure Characterization ◽

Antiprotozoal Activity ◽

Protozoan Parasites ◽

Trypanosoma Brucei Brucei ◽

Antiparasitic Activity ◽

Ic50 Values

1H-3-{4-[(3-Dimethylaminopropyl)aminomethyl]phenyl}-2-phenylindole was synthesized via a multi-step pathway starting from 2-iodoaniline. Structure characterization of this new indole compound was achieved by 1H-NMR, 13C-NMR and ESI-MS spectral analysis. The title compound was screened in vitro against three protozoan parasites (Plasmodium falciparum, Leishmania donovani and Trypanosoma brucei brucei). Biological results showed antiparasitic activity with IC50 values in the μM range.

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Mus81 Endonuclease Localizes to Nucleoli and to Regions of DNA Damage in Human S-phase Cells

Molecular Biology of the Cell ◽

10.1091/mbc.e03-05-0276 ◽

2003 ◽

Vol 14 (12) ◽

pp. 4826-4834 ◽

Cited By ~ 30

Author(s):

Hui Gao ◽

Xiao-Bo Chen ◽

Clare H. McGowan

Keyword(s):

Nuclear Protein ◽

S Phase ◽

Holliday Junctions ◽

Replication Forks ◽

Dna Helicases ◽

Recombination Repair ◽

Higher Eukaryotes ◽

In Cells

Mus81 is a highly conserved substrate specific endonuclease. Human Mus81 cleaves Holliday junctions, replication forks, and 3′ flap substrates in vitro, suggesting a number of possible in vivo functions. We show here that the abundance of human Mus81 peaks in S-phase and remains high in cells that have completed DNA replication and that Mus81 is a predominantly nuclear protein, with super accumulation in nucleoli. Two RecQ related DNA helicases BLM and WRN that are required for recombination repair in human cells colocalize with Mus81 in nucleoli. However, the nucleolar retention of Mus81 is not dependent on the presence of BLM or WRN, or on ongoing transcription. Mus81 is recruited to localized regions of UV damage in S-phase cells, but not in cells that are blocked from replicating DNA or that have completed replication. The retention of human Mus81 at regions of UV-induced damage specifically in S-phase cells suggest that the enzyme is recruited to the sites at which replication forks encounter damaged DNA. The nucleolar concentration of Mus81 suggests that it is required to repair problems that arise most frequently in the highly repetitive nucleolar DNA. Together these data support a role for Mus81 in recombination repair in higher eukaryotes.

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