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Journal articleTiengwe C, Bush PJ, Bangs JD, 2017,
Controlling transferrin receptor trafficking with GPI-valence in bloodstream stage African trypanosomes, PLoS Pathogens, Vol: 13, Pages: 1-24, ISSN: 1553-7366
Bloodstream-formAfricantrypanosomesencodetwostructurallyrelatedglycosylphosphati-dylinositol(GPI)-anchoredproteinsthatarecriticalvirulencefactors,variantsurfaceglyco-protein(VSG)forantigenicvariationandtransferrinreceptor(TfR)forironacquisition.Botharetranscribedfromtheactivetelomericexpressionsite.VSGis a GPI2homodimer;TfRisa GPI1heterodimerof GPI-anchoredESAG6andESAG7.GPI-valencecorrelateswithsecretoryprogressionandfatein bloodstreamtrypanosomes:VSG(GPI2) is a surfacepro-tein;truncatedVSG(GPI0) is degradedin thelysosome;andnativeTfR(GPI1) localizesintheflagellarpocket.Tf:Festarvationresultsin up-regulationandredistributionof TfRto theplasmamembranesuggestinga saturablemechanismforflagellarpocketretention.How-ever,becausesuchsurfaceTfRis non-functionalforligandbindingweproposedthatit rep-resentsGPI2ESAG6homodimersthatareunableto bindtransferrin—therebymimickingnativeVSG.Wenowexploita novelRNAisystemforsimultaneouslethalsilencingof allnativeTfRsubunitsandexclusivein-situexpressionof RNAi-resistant TfRvariantswithvalencesof GPI0–2. Ourresultsconformto thevalencemodel:GPI0ESAG7homodimerstraffickto thelysosomeandGPI2ESAG6homodimersto thecellsurface.However,whenexpressedaloneESAG6is up-regulated~7-fold,leavingtheissueof saturableretentionintheflagellarpocketin question.Therefore,wecreatedanRNAi-resistant GPI2TfRheterodi-merbyfusingtheC-terminaldomainof ESAG6to ESAG7.Co-expressionwithESAG6gen-eratesa functionalheterodimericGPI2TfRthatrestoresTf uptakeandcellviability,andlocalizesto thecellsurface,withoutoverexpression.Theseresultsresolvethelongstandingissueof TfRtraffickingunderover-expressionandconfirmGPIvalenceasa criticaldetermi-nantof intracellularsortingin trypanosomes.
Journal articleWesthorpe F, 2017,
Journal articleTiengwe C, Muratore KA, Bangs JD, 2016,
Variant surface glycoprotein (VSG) is central to antigenic variation in African trypanosomes. Although much prior work documents that VSG is efficiently synthesized and exported to the cell surface, it was recently claimed that 2–3 fold more is synthesized than required, the excess being eliminated by ER‐Associated Degradation (ERAD) (Field et al., 2010). We now reinvestigate VSG turnover and find no evidence for rapid degradation, consistent with a model whereby VSG synthesis is precisely regulated to match requirements for a functional surface coat on each daughter cell. However, using a mutated version of the ESAG7 subunit of the transferrin receptor (E7:Ty) we confirm functional ERAD in trypanosomes. E7:Ty fails to assemble into transferrin receptors and accumulates in the ER, consistent with retention of misfolded protein, and its turnover is selectively rescued by the proteasomal inhibitor MG132. We also show that ER accumulation of E7:Ty does not induce an unfolded protein response. These data, along with the presence of ERAD orthologues in the Trypanosoma brucei genome, confirm ERAD in trypanosomes. We discuss scenarios in which ERAD could be critical to bloodstream parasites, and how these may have contributed to the evolution of antigenic variation in trypanosomes.
Journal articleMarques CA, Tiengwe C, Lemgruber L, et al., 2016,
Diverged composition and regulation of the Trypanosoma brucei origin recognition complex that mediates DNA replication initiation, Nucleic Acids Research, Vol: 44, Pages: 4763-4784, ISSN: 0305-1048
Initiation of DNA replication depends upon recog-nition of genomic sites, termed origins, by AAA+ATPases. In prokaryotes a single factor binds eachorigin, whereas in eukaryotes this role is playedby a six-protein origin recognition complex (ORC).Why eukaryotes evolved a multisubunit initiator, andthe roles of each component, remains unclear. InTrypanosoma brucei, an ancient unicellular eukary-ote, only one ORC-related initiator, TbORC1/CDC6,has been identified by sequence homology. Herewe show that three TbORC1/CDC6-interacting fac-tors also act inT. bruceinuclear DNA replicationand demonstrate that TbORC1/CDC6 interacts in ahigh molecular complex in which a diverged Orc4homologue and one replicative helicase subunit canalso be found. Analysing the subcellular localizationof four TbORC1/CDC6-interacting factors during thecell cycle reveals that one factor, TbORC1B, is nota static constituent of ORC but displays S-phaserestricted nuclear localization and expression, sug-gesting it positively regulates replication. This workshows that ORC architecture and regulation are di-verged features of DNA replication initiation inT. bru-cei, providing new insight into this key stage of eu-karyotic genome copying.
Journal articleTiengwe C, Brown AENA, Bangs JD, 2015,
The unfolded protein response (UPR) is a stress mechanism to cope with misfolded proteins in the early secretory pathway, the hallmark being transcriptional upregulation of endoplasmic reticulum (ER) molecular chaperones such as BiP and protein disulfide isomerase. Despite the lack of transcriptional regulation and the absence of the classical UPR machinery, African trypanosomes apparently respond to persistent ER stress by a UPR-like response, including upregulation of BiP, and a related spliced leader silencing (SLS) response whereby SL RNA transcription is shut down. Initially observed by knockdown of the secretory protein translocation machinery, both responses are also induced by chemical agents known to elicit UPR in mammalian cells (H. Goldshmidt, D. Matas, A. Kabi, A. Carmi, R. Hope, S. Michaeli, PLoS Pathog 6:e1000731, 2010, http://dx.doi.org/10.1371/journal.ppat.1000731). As these findings were generated primarily in procyclic-stage trypanosomes, we have investigated both responses in pathogenic bloodstream-stage parasites. RNA interference (RNAi) silencing of the core translocon subunit Trypanosoma brucei Sec61α (TbSec61α) failed to induce either response. Interestingly, cell growth halted within 16 h of silencing, but sufficient TbSec61α remained to allow full competence for translocation of nascent secretory proteins for up to 24 h, indicating that replication is finely coupled with the capacity to synthesize and transport secretory cargo. Tunicamycin and thapsigargin at concentrations compatible with short-term (4 h) and long-term (24 h) viability also failed to induce any of the indicators of UPR-like or SLS responses. Dithiothreitol (DTT) was lethal at all concentrations tested. These results indicate that UPR-like and SLS responses to persistent ER stress do not occur in bloodstream-stage trypanosomes.
Journal articleKammers K, Cole RN, Tiengwe C, et al., 2015,
We review and demonstrate how an empirical Bayes method, shrinking a protein's sample variance towards a pooled estimate, leads to far more powerful and stable inference to detect significant changes in protein abundance compared to ordinary t-tests. Using examples from isobaric mass labelled proteomic experiments we show how to analyze data from multiple experiments simultaneously, and discuss the effects of missing data on the inference. We also present easy to use open source software for normalization of mass spectrometry data and inference based on moderated test statistics.
Journal articleTiengwe C, Marques CA, McCulloch R, 2014,
Journal articlePovelones ML, Tiengwe C, Gluenz E, et al., 2013,
In an RNAi library screen for loss of kinetoplast DNA (kDNA), we identified an uncharacterized Trypanosoma brucei protein, named TbLOK1, required for maintenance of mitochondrial shape and function. We found the TbLOK1 protein located in discrete patches in the mitochondrial outer membrane. Knock‐down of TbLOK1 in procyclic trypanosomes caused the highly interconnected mitochondrial structure to collapse, forming an unbranched tubule remarkably similar to the streamlined organelle seen in the bloodstream form. Following RNAi, defects in mitochondrial respiration, inner membrane potential and mitochondrial transcription were observed. At later times following TbLOK1 depletion, kDNA was lost and a more drastic alteration in mitochondrial structure was found. Our results demonstrate the close relationship between organelle structure and function in trypanosomes.
Journal articleTiengwe C, Marcello L, Farr H, et al., 2012,
Genome-wide Analysis Reveals Extensive Functional Interaction between DNA Replication Initiation and Transcription in the Genome of Trypanosoma brucei, Cell Reports, Vol: 2, Pages: 185-197, ISSN: 2211-1247
Identification of replication initiation sites, termed origins, is a crucial step in understanding genome transmission in any organism. Transcription of the Trypanosoma brucei genome is highly unusual, with each chromosome comprising a few discrete transcription units. To understand how DNA replication occurs in the context of such organization, we have performed genome-wide mapping of the binding sites of the replication initiator ORC1/CDC6 and have identified replication origins, revealing that both localize to the boundaries of the transcription units. A remarkably small number of active origins is seen, whose spacing is greater than in any other eukaryote. We show that replication and transcription in T. brucei have a profound functional overlap, as reducing ORC1/CDC6 levels leads to genome-wide increases in mRNA levels arising from the boundaries of the transcription units. In addition, ORC1/CDC6 loss causes derepression of silent Variant Surface Glycoprotein genes, which are critical for host immune evasion.
Journal articleTiengwe C, Marcello L, Farr H, et al., 2012,
Identification of ORC1/CDC6-Interacting Factors in Trypanosoma brucei Reveals Critical Features of Origin Recognition Complex Architecture, PLoS ONE, Vol: 7, ISSN: 1932-6203
DNA Replication initiates by formation of a pre-replication complex on sequences termed origins. In eukaryotes, the pre-replication complex is composed of the Origin Recognition Complex (ORC), Cdc6 and the MCM replicative helicase inconjunction with Cdt1. Eukaryotic ORC is considered to be composed of six subunits, named Orc1–6, and monomeric Cdc6is closely related in sequence to Orc1. However, ORC has been little explored in protists, and only a single ORC protein,related to both Orc1 and Cdc6, has been shown to act in DNA replication inTrypanosoma brucei. Here we identify threehighly diverged putativeT. bruceiORC components that interact with ORC1/CDC6 and contribute to cell division. Two ofthese factors are so diverged that we cannot determine if they are eukaryotic ORC subunit orthologues, or are parasite-specific replication factors. The other we show to be a highly diverged Orc4 orthologue, demonstrating that this is one ofthe most widely conserved ORC subunits in protists and revealing it to be a key element of eukaryotic ORC architecture.Additionally, we have examined interactions amongst theT. bruceiMCM subunits and show that this has the conventionaleukaryotic heterohexameric structure, suggesting that divergence in theT. bruceireplication machinery is limited to theearliest steps in origin licensing.
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