Identification of a small-molecule inhibitor that selectively blocks DNA-binding by Trypanosoma brucei replication protein A1.

Replication Protein A (RPA) is a broadly conserved complex comprised of the RPA1, 2 and 3 subunits. RPA protects the exposed single-stranded DNA (ssDNA) during DNA replication and repair. Using structural modeling, we discover an inhibitor, JC-229, that targets RPA1 in Trypanosoma brucei, the causative parasite of African trypanosomiasis.

UMSBP2 is chromatin remodeler that functions in regulation of gene expression and suppression of antigenic variation in trypanosomes.

Universal Minicircle Sequence binding proteins (UMSBPs) are CCHC-type zinc-finger proteins that bind the single-stranded G-rich UMS sequence, conserved at the replication origins of minicircles in the kinetoplast DNA, the mitochondrial genome of kinetoplastids. Trypanosoma brucei UMSBP2 has been recently shown to colocalize with telomeres and to play an essential role in chromosome end protection. Here we report that TbUMSBP2 decondenses in vitro DNA molecules, which were condensed by core histones H2B, H4 or linker histone H1.

Functional insights from a surface antigen mRNA-bound proteome.

is the causative agent of human sleeping sickness. The parasites' variant surface glycoprotein (VSG) enables them to evade adaptive immunity via antigenic variation. VSG comprises 10% of total cell protein and the high stability of VSG mRNA is essential for trypanosome survival.

Insights into the functions and RNA binding of ZC3H22, RBP9 and DRBD7.

Trypanosoma brucei is unusually reliant on mRNA-binding proteins to control mRNA fate, because its protein-coding genes lack individual promoters. We here focus on three trypanosome RNA-binding proteins. ZC3H22 is specific to Tsetse fly forms, RBP9 is preferentially expressed in bloodstream forms; and DRBD7 is constitutively expressed.

A potential role for a novel ZC3H5 complex in regulating mRNA translation in .

In and related kinetoplastids, gene expression regulation occurs mostly posttranscriptionally. Consequently, RNA-binding proteins play a critical role in the regulation of mRNA and protein abundance. Yet, the roles of many RNA-binding proteins are not understood.

The Tethering Assay: A Simple Method for the Characterization of mRNA-Fate Regulators.

In trypanosomatids, posttranscriptional controls are very important in regulation of individual gene expression. These are achieved through combinatorial sets of RNA-binding proteins (RBPs) which recognize RNA regulatory motifs or regions of secondary structure within RNAs. To analyze the potential functional impact of an RBP on their mRNA targets, we have applied a robust technique called tethering assay.

Identifying Trypanosome Protein-RNA Interactions Using RIP-Seq.

Trypanosomatids rely primarily on posttranscriptional mechanisms for the control of gene expression, with regulation of RNA processing, localization, degradation, and translation by RNA-binding proteins (RBPs). To determine the mechanisms by which RBPs control gene expression in trypanosomatids, transcriptome-wide identification of mRNA targets and mapping of the RNA-binding site is required. Here we present our most current RIP-Seq (RNA immunoprecipitation followed by high-throughput sequencing) protocol that we generally apply to elucidate RNA/protein interactions in Trypanosoma brucei.

The endoplasmic reticulum-associated mRNA-binding proteins ERBP1 and ERBP2 interact in bloodstream-form .

Kinetoplastids rely heavily on post-transcriptional mechanisms for control of gene expression, and on RNA-binding proteins that regulate mRNA splicing, translation and decay. ERBP1 (Tb927.10.

Mechanistic Similarities between Antigenic Variation and Antibody Diversification during Trypanosoma brucei Infection.

Trypanosoma brucei, which causes African trypanosomiasis, avoids immunity by periodically switching its surface composition. The parasite is coated by 10 million identical, monoallelically expressed variant surface glycoprotein (VSG) molecules. Multiple distinct parasites (with respect to their VSG coat) coexist simultaneously during each wave of parasitemia.

Codon Usage in Trypanosomatids: The Bias of Expression.

Translation and RNA decay, two processes in which all mRNAs are engaged, are intimately related processes. Two new studies demonstrate that, in trypanosomatids, codon usage largely shapes mRNA abundance in a translation-dependent manner. The findings indicate that mRNA decay control by codon choice is an ancient and conserved mechanism.

High-throughput Methods for Dissection of Trypanosome Gene Regulatory Networks.

Unlabelled: From synthesis to decay, mRNA associates with RNA-binding proteins (RBPs) establishing dynamic ribonucleoprotein particles (RNPs). Understanding the composition and function of RNPs is fundamental to understanding how eukaryotic mRNAs are controlled. This is especially relevant for trypanosomes and related kinetoplastid parasites, which mostly rely on post-transcriptional mechanisms to control gene expression.

Gene expression regulatory networks in Trypanosoma brucei: insights into the role of the mRNA-binding proteome.

Control of gene expression at the post-transcriptional level is essential in all organisms, and RNA-binding proteins play critical roles from mRNA synthesis to decay. To fully understand this process, it is necessary to identify the complete set of RNA-binding proteins and the functional consequences of the protein-mRNA interactions. Here, we provide an overview of the proteins that bind to mRNAs and their functions in the pathogenic bloodstream form of Trypanosoma brucei.

Drug target identification using a trypanosome overexpression library.

Elucidation of molecular targets is very important for lead optimization during the drug development process. We describe a direct method to find targets of antitrypanosomal compounds against Trypanosoma brucei using a trypanosome overexpression library. As proof of concept, we treated the library with difluoromethylornithine and DDD85646 and identified their respective targets, ornithine decarboxylase and N-myristoyltransferase.

A genome-wide tethering screen reveals novel potential post-transcriptional regulators in Trypanosoma brucei.

In trypanosomatids, gene expression is regulated mainly by post-transcriptional mechanisms, which affect mRNA processing, translation and degradation. Currently, our understanding of factors that regulate either mRNA stability or translation is rather limited. We know that often, the regulators are proteins that bind to the 3'-untranslated region; they presumably interact with ribonucleases and translation factors.

Trypanosome MKT1 and the RNA-binding protein ZC3H11: interactions and potential roles in post-transcriptional regulatory networks.

The trypanosome zinc finger protein ZC3H11 binds to AU-rich elements in mRNAs. It is essential for survival of the mammalian-infective bloodstream form, where it stabilizes several mRNAs including some encoding chaperones, and is also required for stabilization of chaperone mRNAs during the heat-shock response in the vector-infective procyclic form. When ZC3H11 was artificially 'tethered' to a reporter mRNA in bloodstream forms it increased reporter expression.

The CAF1-NOT complex of trypanosomes.

In African trypanosomes, there is no control of transcription initiation by RNA polymerase II at the level of individual protein-coding genes. Transcription is polycistronic, and individual mRNAs are excised by trans-splicing and polyadenylation. As a consequence, trypanosomes are uniquely reliant on post-transcriptional mechanisms for control of gene expression.

Bordetella pertussis iron regulated proteins as potential vaccine components.

Bordetella pertussis is the etiologic agent of whooping cough, an illness whose incidence has been increasing over the last decades. Pertussis reemergence despite high vaccination coverage, together with the recent isolation of circulating strains deficient in some of the vaccine antigens, highlight the need for new vaccines. Proteins induced under physiological conditions, such as those required for nutrient acquisition during infection, might represent good targets for better preventive strategies.

Trypanosome CNOT10 is essential for the integrity of the NOT deadenylase complex and for degradation of many mRNAs.

The degradation of most eukaryotic mRNAs is initiated by removal of the poly(A) tail, and the major deadenylase activity is associated with the CCR4/CAF1/NOT complex (NOT complex). We here study the role of CNOT10, a protein that is found in human and trypanosome, but not in yeast, NOT complexes. Trypanosome (Tb) CNOT10 is essential for growth.

Trypanosomatid pin1-type peptidyl-prolyl isomerase is cytosolic and not essential for cell proliferation.

Pin1-type peptidyl-prolyl cis/trans isomerases (PPIases) isomerise the peptide bond of specific phosphorylated (Ser/Thr)-Pro residues, regulating various cellular events. Previously, we reported a Pin1-type PPIase in Trypanosoma cruzi, but little is known about its function and subcellular localization. Immunofluorescence analysis revealed that in contrast with Pin1-like proteins from diverse organisms, TcPin1 mainly localized in the cytoplasm and was excluded from the nuclei.

Identification of a new protective antigen of Bordetella pertussis.

Antigenic proteins whose expression is induced under iron starvation, an environmental condition that bacterial pathogens have to face during colonization, might be potential candidates for improved vaccine. By mean of immune proteomics we identified novel antigens of Bordetella pertussis maximally expressed under iron limitation. Among them, Bp1152 (named as IRP1-3) showed a particularly strong reaction with human IgG purified from pooled sera of pertussis-infected individuals.

Functional characterization of methionine sulfoxide reductase A from Trypanosoma spp.

Methionine is an amino acid susceptible to being oxidized to methionine sulfoxide (MetSO). The reduction of MetSO to methionine is catalyzed by methionine sulfoxide reductase (MSR), an enzyme present in almost all organisms. In trypanosomatids, the study of antioxidant systems has been mainly focused on the involvement of trypanothione, a specific redox component in these organisms.

Identification of an atypical peptidyl-prolyl cis/trans isomerase from trypanosomatids.

The parvulin family of peptidyl-prolyl cis/trans isomerases (PPIases) catalyzes the cis/trans isomerization of the peptide bonds preceding Pro residues. Eukaryotic parvulin-type PPIases have been shown to be involved in cell proliferation and cell cycle progression. Here we present the biochemical and molecular characterization of a novel multi-domain parvulin-type PPIase from the human pathogenic Trypanosoma cruzi, annotated as TcPar45.

Identification of regions critically affecting kinetics and allosteric regulation of the Escherichia coli ADP-glucose pyrophosphorylase by modeling and pentapeptide-scanning mutagenesis.

ADP-glucose pyrophosphorylase (ADP-Glc PPase) is the enzyme responsible for the regulation of bacterial glycogen synthesis. To perform a structure-function relationship study of the Escherichia coli ADP-Glc PPase enzyme, we studied the effects of pentapeptide insertions at different positions in the enzyme and analyzed the results with a homology model. We randomly inserted 15 bp in a plasmid with the ADP-Glc PPase gene.

The Trypanosoma cruzi PIN1 gene encodes a parvulin peptidyl-prolyl cis/trans isomerase able to replace the essential ESS1 in Saccharomyces cerevisiae.

Parvulins are a conserved group of peptidyl-prolyl cis/trans isomerases (PPIases) that catalyze the cis/trans isomerization of proline-preceding peptide bonds. Parvulin-class PPIases are structurally unrelated to cyclophilins and FK506-binding proteins that are defined as receptors for immunosuppressive drugs. In Trypanosoma cruzi we identified parvulin TcPIN1 as a homolog of the human hPin1 PPIase.