Phosphoproteomic analysis of mammalian infective Trypanosoma brucei subjected to heat shock suggests atypical mechanisms for thermotolerance

The symptoms of African sleeping sickness, caused by the parasite Trypanosoma brucei, can include periods of fever as high as 41 °C which triggers a heat shock response in the parasite. To capture events involved in sensing and responding to heat shock in the mammalian infective form we have conducted a SILAC-based quantitative proteomic and phosphoproteomic analysis of T. brucei cells treated at 41 °C for 1h. Our analysis identified 193 heat shock responsive phosphorylation sites with an average of 5-fold change in abundance, but only 20 heat shock responsive proteins with average of 1.5-fold change. These data indicate that protein abundance does not rapidly respond (≤1 h) to heat shock, and that the changes observed in phosphorylation site abundance are larger and more widespread. The heat shock responsive phosphorylation sites showed enrichment of RNA binding proteins with putative roles in heat shock response included P-body / stress granules and the eukaryotic translation initiation 4F complex. The ZC3H11-MKT1 complex, which stabilises mRNAs of thermotolerance proteins, appears to represent a key signal integration node in the heat shock response

Transcriptome analysis of differentiating trypanosomes reveals the existence of multiple post-transcriptional regulons

<p>Abstract</p> <p>Background</p> <p>Trypanosome gene expression is regulated almost exclusively at the post-transcriptional level, with mRNA degradation playing a decisive role. When trypanosomes are transferred from the blood of a mammal to the midgut of a Tsetse fly, they transform to procyclic forms: gene expression is reprogrammed, changing the cell surface and switching the mode of energy metabolism. Within the blood, trypanosomes can pre-adapt for Tsetse transmission, becoming growth-arrested stumpy forms. We describe here the transitions in gene expression that occur during differentiation of <it>in-vitro </it>cultured bloodstream forms to procyclic forms.</p> <p>Results</p> <p>Some mRNAs showed changes within 30 min of <it>cis-</it>aconitate addition, whereas others responded 12-24 hours later. For the first 12 h after addition of <it>cis</it>-aconitate, cells accumulated at the G1 phase of the cell cycle, and showed decreases in mRNAs required for proliferation, mimicking the changes seen in stumpy forms: many mRNAs needed for ribosomal and flagellar biogenesis showed striking co-regulation. Other mRNAs encoding components of signal transduction pathways and potential regulators were specifically induced only during differentiation. Messenger RNAs encoding proteins required for individual metabolic pathways were often co-regulated.</p> <p>Conclusion</p> <p>Trypanosome genes form post-transcriptional regulons in which mRNAs with functions in particular pathways, or encoding components of protein complexes, show almost identical patterns of regulation.</p

Roles of the Nfu Fe-S targeting factors in the trypanosome mitochondrion

Iron–sulphur clusters (ISCs) are protein co-factors essential for a wide range of cellular functions. The core iron–sulphur cluster assembly machinery resides in the mitochondrion, yet due to export of an essential precursor from the organelle, it is also needed for cytosolic and nuclear iron–sulphur cluster assembly. In mitochondria all [4Fe–4S] iron–sulphur clusters are synthesised and transferred to specific apoproteins by so-called iron–sulphur cluster targeting factors. One of these factors is the universally present mitochondrial Nfu1, which in humans is required for the proper assembly of a subset of mitochondrial [4Fe–4S] proteins. Although most eukaryotes harbour a single Nfu1, the genomes of Trypanosoma brucei and related flagellates encode three Nfu genes. All three Nfu proteins localise to the mitochondrion in the procyclic form of T. brucei, and TbNfu2 and TbNfu3 are both individually essential for growth in bloodstream and procyclic forms, suggesting highly specific functions for each of these proteins in the trypanosome cell. Moreover, these two proteins are functional in the iron–sulphur cluster assembly in a heterologous system and rescue the growth defect of a yeast deletion mutant

Cell cycle synchronisation of Trypanosoma brucei by centrifugal counter-flow elutriation reveals the timing of nuclear and kinetoplast DNA replication

We report an optimised centrifugal counter-flow elutriation protocol for the rapid and direct isolation of G1 cell cycle synchronised populations of both the procyclic and bloodstream form stages of Trypanosoma brucei that yields viable and proliferative cells. The high quality of the synchronisation achieved can be judged by the uniform DNA content, narrow size distribution, synchronous division, and the maintenance of synchronicity into subsequent cell cycles. We show that early-eluting fractions represent different G1 subpopulations that progress through the cell cycle with distinct temporal profiles post-elutriation, as exemplified by the observation of the maturation of a second flagellar basal body in late G1 phase, DNA replication in S phase, and dimethylation of histone H3 in mitosis/cytokinesis. We use our temporal observations to construct a revised model of the relative timing and duration of the nuclear and kinetoplast cell cycle that differs from the current model

Extensive translational regulation through the proliferative transition of Trypanosoma cruzi revealed by Multi-Omics

Trypanosoma cruzi is the etiological agent for Chagas disease, a neglected parasitic disease in Latin America. Gene transcription control governs the eukaryotic cell replication but is absent in trypanosomatids; thus, it must be replaced by posttranscriptional regulatory events. We investigated the entrance into the T. cruzi replicative cycle using ribosome profiling and proteomics on G1/S epimastigote cultures synchronized with hydroxyurea. We identified 1,784 translationally regulated genes (change > 2, false-discovery rate [FDR]  1.5, FDR < 0.05), respectively. A major translational remodeling accompanied by an extensive proteome change is found, while the transcriptome remains largely unperturbed at the replicative entrance of the cell cycle. The differentially expressed genes comprise specific cell cycle processes, confirming previous findings while revealing candidate cell cycle regulators that undergo previously unnoticed translational regulation. Clusters of genes showing a coordinated regulation at translation and protein abundance share related biological functions such as cytoskeleton organization and mitochondrial metabolism; thus, they may represent posttranscriptional regulons. The translatome and proteome of the coregulated clusters change in both coupled and uncoupled directions, suggesting that complex cross talk between the two processes is required to achieve adequate protein levels of different regulons. This is the first simultaneous assessment of the transcriptome, translatome, and proteome of trypanosomatids, which represent a paradigm for the absence of transcriptional control. The findings suggest that gene expression chronology along the T. cruzi cell cycle is controlled mainly by translatome and proteome changes coordinated using different mechanisms for specific gene groups

Dynamic regulation of the Trypanosoma brucei transferrin receptor in response to iron starvation is mediated via the 3’UTR

The bloodstream form of the parasite Trypanosoma brucei obtains iron from its mammalian host by receptor-mediated endocytosis of host transferrin through its own unique transferrin receptor (TbTfR). Expression of TbTfR rapidly increases upon iron starvation by post-transcriptional regulation through a currently undefined mechanism that is distinct from the mammalian iron response system. We have created reporter cell lines by fusing the TbTfR 3’UTR or a control Aldolase 3’UTR to reporter genes encoding GFP or firefly Luciferase, and inserted the fusions into a bloodstream form cell line at a tagged ribosomal RNA locus. Fusion of the TbTfR 3’UTR is sufficient to significantly repress the expression of the reporter proteins under normal growth conditions. Under iron starvation conditions we observed upregulation of the mRNA and protein level of the TbTfR 3’UTR fusions only, with a magnitude and timing consistent with that reported for upregulation of the TbTfR. We conclude that the dynamic regulation of the T. brucei transferrin receptor in response to iron starvation is mediated via its 3’UTR, and that the effect is independent of genomic location

Early diagnosis of acute myocardial infarction in patients with pre-existing coronary artery disease using more sensitive cardiac troponin assays

Aims We sought to examine the diagnostic and prognostic utility of sensitive cardiac troponin (cTn) assays in patients with pre-existing coronary artery disease (CAD). Methods and results We conducted a multicentre study to examine the diagnostic accuracy of one high-sensitive and two sensitive cTn assays in 1098 consecutive patients presenting with symptoms suggestive of acute myocardial infarction (AMI), of whom 401 (37%) had pre-existing CAD. Measurements of Roche high-sensitive cTnT (hs-cTnT), Siemens cTnI-Ultra, Abbott-Architect cTnI and the standard assay (Roche cTnT) were performed in a blinded fashion. The final diagnosis was adjudicated by two independent cardiologists. Acute myocardial infarction was the final diagnosis in 19% of CAD patients. Among patients with diagnoses other than AMI, baseline cTn levels were elevated above the 99th percentile with Roche hs-cTnT in 40%, with Siemens TnI-Ultra in 15%, and Abbott-Architect cTnI in 13% of them. In patients with pre-existing CAD, the diagnostic accuracy at presentation, quantified by the area under the receiver operator characteristic curve (AUC), was significantly greater for the sensitive cTn assays compared with the standard assay (AUC for Roche hs-cTnT, 0.92; Siemens cTnI-Ultra, 0.94; and Abbott-Architect cTnI, 0.93 vs. AUC for the standard assay, 0.87; P < 0.01 for all comparisons). Elevated levels of cTn measured with the sensitive assays predicted mortality irrespective of pre-existing CAD, age, sex, and cardiovascular risk factors. Conclusion Sensitive cTn assays have high-diagnostic accuracy also in CAD patients. Mild elevations are common in non-AMI patients and test-specific optimal cut-off levels tend to be higher in CAD patients than in patients without history of CAD. Sensitive cTn assays also retain prognostic value. (ClinicalTrials.gov number, NCT00470587

Cytokinesis in bloodstream stage Trypanosoma brucei requires a family of katanins and spastin

Microtubule severing enzymes regulate microtubule dynamics in a wide range of organisms and are implicated in important cell cycle processes such as mitotic spindle assembly and disassembly, chromosome movement and cytokinesis. Here we explore the function of several microtubule severing enzyme homologues, the katanins (KAT80, KAT60a, KAT60b and KAT60c), spastin (SPA) and fidgetin (FID) in the bloodstream stage of the African trypanosome parasite, Trypanosoma brucei. The trypanosome cytoskeleton is microtubule based and remains assembled throughout the cell cycle, necessitating its remodelling during cytokinesis. Using RNA interference to deplete individual proteins, we show that the trypanosome katanin and spastin homologues are non-redundant and essential for bloodstream form proliferation. Further, cell cycle analysis revealed that these proteins play essential but discrete roles in cytokinesis. The KAT60 proteins each appear to be important during the early stages of cytokinesis, while downregulation of KAT80 specifically inhibited furrow ingression and SPA depletion prevented completion of abscission. In contrast, RNA interference of FID did not result in any discernible effects. We propose that the stable microtubule cytoskeleton of T. brucei necessitates the coordinated action of a family of katanins and spastin to bring about the cytoskeletal remodelling necessary to complete cell divisio

A study on the functions of ubiquitin metabolic system related gene FBG2 in gastric cancer cell line

<p>Abstract</p> <p>Background</p> <p>FBG2 (F-BOX6) gene is an important member in ubiquitin metabolic system F-BOX family, and forms E3 complex with the other members in the family. But its role in gastric cancer is still not clear. In the present study, we intended to investigate the influence of FBG2 on the growth, proliferation, apoptosis, invasion and cell cycle of the gastric cancer line MKN45 and gastric cell line HFE145.</p> <p>Methods</p> <p>As a critical component of ubiquitin-protein ligase complex, FBG2 cDNA was subcloned into a constitutive vector PCDNA3.1 followed by transfection in MKN45 and HFE145 by using liposome. Then stable transfectants were selected and appraised. The apoptosis and cell cycles of these clones were analyzed by using flow cytometry. The growth and proliferation were analyzed by cell growth curves and colony-forming assay respectively. The invasion of these clones was tested by using cancer cell migration assay. The FBG2 stable expression clones(MKN-FBG2 and HFE-FBG2) and their control groups were detected and compared respectively.</p> <p>Results</p> <p>MKN-FBG2 grew faster than MKN45 and MKN-PC(MKN45 transfected with PCDNA3.1 vector). HFE-FBG2 grew faster than HFE145 and HFE-PC(HFE145 transfected with PCDNA3.1 vector). The cell counts of MKN-FBG2 in the forth, fifth, sixth and seventh days were significantly more than those of others (P < 0.05). Cell cycle analysis showed that MKN-FBG2 and HFE-FBG2 proliferated faster, proportions of cells in G2-M and S were different significantly with control groups (P < 0.05). Results of colony-forming assay showed that the colony formation rates of MKN-FBG2 and HFE-FBG2 were higher than those of control groups (P < 0.05). The results of cell migration assay were all negative.</p> <p>Conclusion</p> <p>FBG2 can promote the growth and proliferation of gastric cancer cells and normal gastric cells. It can help tumor cell maintain malignant phenotype too. But it can have a negative influence on the apoptosis or the ability of invasion of gastric cancer cells.</p

Identification and functional characterisation of CRK12:CYC9, a novel cyclin-dependent kinase (CDK)-cyclin complex in Trypanosoma brucei

The protozoan parasite, Trypanosoma brucei, is spread by the tsetse fly and causes trypanosomiasis in humans and animals. Both the life cycle and cell cycle of the parasite are complex. Trypanosomes have eleven cdc2-related kinases (CRKs) and ten cyclins, an unusually large number for a single celled organism. To date, relatively little is known about the function of many of the CRKs and cyclins, and only CRK3 has previously been shown to be cyclin-dependent in vivo. Here we report the identification of a previously uncharacterised CRK:cyclin complex between CRK12 and the putative transcriptional cyclin, CYC9. CRK12:CYC9 interact to form an active protein kinase complex in procyclic and bloodstream T. brucei. Both CRK12 and CYC9 are essential for the proliferation of bloodstream trypanosomes in vitro, and we show that CRK12 is also essential for survival of T. brucei in a mouse model, providing genetic validation of CRK12:CYC9 as a novel drug target for trypanosomiasis. Further, functional characterisation of CRK12 and CYC9 using RNA interference reveals roles for these proteins in endocytosis and cytokinesis, respectively