Medical Biology - Research Publications
Permanent URI for this collection
Search Results
1 - 3 of 3
-
ItemNo Preview AvailableMultimodal regulation of encystation in Giardia duodenalis revealed by deep proteomics(ELSEVIER SCI LTD, 2021-09)Cyst formation in the parasitic protist Giardia duodenalis is critical to its transmission. Existing proteomic data quantifies only 17% of coding genes transcribed during encystation and does not cover the complete process from trophozoite to mature cyst. Using high-resolution mass spectrometry, we have quantified proteomic changes across encystation and compared this with published transcriptomic data. We reproducibly identified 3863 (64.5% of Giardia proteins) and quantified 3382 proteins (56.5% of Giardia proteins) over standard trophozoite growth (TY), during low-bile encystation priming (LB), 16 h into encystation (EC), and at cyst maturation (C). This work provides the first known expanded observation of encystation at the proteomic level and triples the coverage of previous encystation proteomes. One-third (1169 proteins) of the quantified proteome is differentially expressed in the mature cyst relative to the trophozoite, including proteasomal machinery, metabolic pathways, and secretory proteins. Changes in lipid metabolism indicated a shift in lipid species dependency during encystation. Consistent with this, we identified the first, putative lipid transporters in this species, representing the steroidogenic acute regulatory protein-related lipid transfer (StARkin), oxysterol binding protein related protein (ORP/Osh) and glycosphingolipid transfer protein (GLTP) families, and follow their differential expression over cyst formation. Lastly, we undertook correlation analyses of the transcriptome and proteome of trophozoites and cysts, and found evidence of post-transcriptional regulation of key protein classes (RNA binding proteins) and stage-specific genes (encystation markers) implicating translation-repression in encystation. We provide the most extensive proteomic analysis of encystation in Giardia to date and the first known exploration across its complete duration. This work identifies encystation as highly coordinated, involving major changes in proteostasis, metabolism and membrane dynamics, and indicates a potential role for post-transcriptional regulation, mediated through RNA-binding proteins. Together our work provides a valuable resource for Giardia research and the development of transmission-blocking anti-giardials.
-
ItemNo Preview AvailableRecent advances in functional research in Giardia intestinalis(ELSEVIER ACADEMIC PRESS INC, 2020-01-01)This review considers current advances in tools to investigate the functional biology of Giardia, it's coding and non-coding genes, features and cellular and molecular biology. We consider major gaps in current knowledge of the parasite and discuss the present state-of-the-art in its in vivo and in vitro cultivation. Advances in in silico tools, including for the modelling non-coding RNAs and genomic elements, as well as detailed exploration of coding genes through inferred homology to model organisms, have provided significant, primary level insight. Improved methods to model the three-dimensional structure of proteins offer new insights into their function, and binding interactions with ligands, other proteins or precursor drugs, and offer substantial opportunities to prioritise proteins for further study and experimentation. These approaches can be supplemented by the growing and highly accessible arsenal of systems-based methods now being applied to Giardia, led by genomic, transcriptomic and proteomic methods, but rapidly incorporating advanced tools for detection of real-time transcription, evaluation of chromatin states and direct measurement of macromolecular complexes. Methods to directly interrogate and perturb gene function have made major leaps in recent years, with CRISPr-interference now available. These approaches, coupled with protein over-expression, fluorescent labelling and in vitro and in vivo imaging, are set to revolutionize the field and herald an exciting time during which the field may finally realise Giardia's long proposed potential as a model parasite and eukaryote.
-
ItemEukaryote-ConservedMethylarginine Is Absent in Diplomonads and Functionally Compensated in Giardia(OXFORD UNIV PRESS, 2020-12)Methylation is a common posttranslational modification of arginine and lysine in eukaryotic proteins. Methylproteomes are best characterized for higher eukaryotes, where they are functionally expanded and evolved complex regulation. However, this is not the case for protist species evolved from the earliest eukaryotic lineages. Here, we integrated bioinformatic, proteomic, and drug-screening data sets to comprehensively explore the methylproteome of Giardia duodenalis-a deeply branching parasitic protist. We demonstrate that Giardia and related diplomonads lack arginine-methyltransferases and have remodeled conserved RGG/RG motifs targeted by these enzymes. We also provide experimental evidence for methylarginine absence in proteomes of Giardia but readily detect methyllysine. We bioinformatically infer 11 lysine-methyltransferases in Giardia, including highly diverged Su(var)3-9, Enhancer-of-zeste and Trithorax proteins with reduced domain architectures, and novel annotations demonstrating conserved methyllysine regulation of eukaryotic elongation factor 1 alpha. Using mass spectrometry, we identify more than 200 methyllysine sites in Giardia, including in species-specific gene families involved in cytoskeletal regulation, enriched in coiled-coil features. Finally, we use known methylation inhibitors to show that methylation plays key roles in replication and cyst formation in this parasite. This study highlights reduced methylation enzymes, sites, and functions early in eukaryote evolution, including absent methylarginine networks in the Diplomonadida. These results challenge the view that arginine methylation is eukaryote conserved and demonstrate that functional compensation of methylarginine was possible preceding expansion and diversification of these key networks in higher eukaryotes.