Browsing by Author "Lucas, Olivier"

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Coordinated progression through two subtranscriptomes underlies the tachyzoitecycle of Toxoplasma gondii
(2010-08) Behnke, Michael S.; Wootton, John C.; Lehmann, Margaret M.; Radke, Josh B.; Lucas, Olivier; Nawas, Julie; Sibley, L. David; White, Michael W.
Background Apicomplexan parasites replicate by varied and unusual processes where the typically eukaryotic expansion of cellular components and chromosome cycle are coordinated with the biosynthesis of parasite-specific structures essential for transmission. Methodology/Principal Findings Here we describe the global cell cycle transcriptome of the tachyzoite stage of Toxoplasma gondii. In dividing tachyzoites, more than a third of the mRNAs exhibit significant cyclical profiles whose timing correlates with biosynthetic events that unfold during daughter parasite formation. These 2,833 mRNAs have a bimodal organization with peak expression occurring in one of two transcriptional waves that are bounded by the transition into S phase and cell cycle exit following cytokinesis. The G1-subtranscriptome is enriched for genes required for basal biosynthetic and metabolic functions, similar to most eukaryotes, while the S/M-subtranscriptome is characterized by the uniquely apicomplexan requirements of parasite maturation, development of specialized organelles, and egress of infectious daughter cells. Two dozen AP2 transcription factors form a series through the tachyzoite cycle with successive sharp peaks of protein expression in the same timeframes as their mRNA patterns, indicating that the mechanisms responsible for the timing of protein delivery might be mediated by AP2 domains with different promoter recognition specificities. Conclusion/Significance Underlying each of the major events in apicomplexan cell cycles, and many more subordinate actions, are dynamic changes in parasite gene expression. The mechanisms responsible for cyclical gene expression timing are likely crucial to the efficiency of parasite replication and may provide new avenues for interfering with parasite growth.
Cytoprotective Nrf2 pathway is induced in chronically txnrd 1-deficient hepatocytes
(2009-07) Suvorova, Elena S.; Lucas, Olivier; Weisend, Carla M.; Rollins, MaryClare F.; Merrill, Gary F.; Capecchi, Mario R.; Schmidt, Edward E.
"Background Metabolically active cells require robust mechanisms to combat oxidative stress. The cytoplasmic thioredoxin reductase/thioredoxin (Txnrd1/Txn1) system maintains reduced protein dithiols and provides electrons to some cellular reductases, including peroxiredoxins. Principal Findings Here we generated mice in which the txnrd1 gene, encoding Txnrd1, was specifically disrupted in all parenchymal hepatocytes. Txnrd1-deficient livers exhibited a transcriptome response in which 56 mRNAs were induced and 12 were repressed. Based on the global hybridization profile, this represented only 0.3% of the liver transcriptome. Since most liver mRNAs were unaffected, compensatory responses were evidently effective. Nuclear pre-mRNA levels indicated the response was transcriptional. Twenty-one of the induced genes contained known antioxidant response elements (AREs), which are binding sites for the oxidative and chemical stress-induced transcription factor Nrf2. Txnrd1-deficient livers showed increased accumulation of nuclear Nrf2 protein and chromatin immunoprecipitation on the endogenous nqo1 and aox1 promoters in fibroblasts indicated that Txnrd1 ablation triggered in vivo assembly of Nrf2 on each. Conclusions Chronic deletion of Txnrd1 results in induction of the Nrf2 pathway, which contributes to an effective compensatory response."
Molecular and systemic functions of the vertebrate-specific TATA-binding protein N terminus
(Montana State University - Bozeman, College of Agriculture, 2009) Lucas, Olivier; Chairperson, Graduate Committee: Edward E. Schmidt; Michael W. White (co-chair)
The invertebrate/vertebrate transition and associated innovations can be regarded as a major event in evolution. Recent molecular progresses invite to an analysis of the events leading to the apparition of vertebrates and the underlying embellishment in gene regulation. In eukaryotes, the TATA-Binding Protein (TBP) has a central role in transcription initiation of most genes. TBP is comprised of a highly conserved DNA binding domain and, in vertebrates, it also contains a novel region: the N-terminal TBP protein coding sequence. The role of the TBP-N is largely unknown, but previous studies suggest that it is important for fetal survival. Most animals lacking the TBP-N (tbp Delta N/Delta N) die before weaning. The goal of the present work was to establish a deeper knowledge of the vertebrate-specific TBP-N. It was hypothesized that TBP-N could be involved in protein-protein interactions and that the high degree of similarity of TBP-N protein sequences in different species could correlate with similar functions. To test those hypotheses, two independent approaches were taken: (1) Protein-protein interactions involving the TBP-N via unbiased screens were characterized. (2) The mouse TBP-N was replaced by a similar and homologous TBP-N, in vivo, through homologous recombination. The TBP-N-replacement mutation was characterized through pathway analyses, bioinformatics, and whole-animal physiology. Screens for proteins interacting with the TBP-N of hagfish (hf), a basal vertebrate, uncovered hfPitxA. The Pitx family of transcription factors are proteins important in vertebrate development. The mouse paralogs of hfPitxA, Pitx1 and Pitx2, were found to interact with the mouse TBP-N. Moreover, the interaction appeared functional as it regulated the expression of nppa, a known target gene of Pitx2. In vivo replacement of the mouse TBP-N with the similar hfTBP-N did not affect the survival. Gene expression analysis indicated that lipid metabolism pathways were affected in animals lacking the TBP-N or when the hfTBP-N was present. Further analyses pointed toward a potential defect in insulin response and an abnormal hepatic fat storage. The data presented here argues in favor of an important role for TBP-N in vertebrate-specific gene regulation. More specifically, it is likely involved in heart development and in regulation of lipid metabolism.
A systematic screen to discover and analyze apicoplast proteins identifies a conserved and essential protein import factor
(2011-12) Sheiner, Lilach; Demerly, Jessica L.; Poulsen, Nicole; Beatty, Wandy L.; Lucas, Olivier; Behnke, Michael S.; White, Michael W.; Striepen, Boris
Parasites of the phylum Apicomplexa cause diseases that impact global health and economy. These unicellular eukaryotes possess a relict plastid, the apicoplast, which is an essential organelle and a validated drug target. However, much of its biology remains poorly understood, in particular its elaborate compartmentalization: four membranes defining four different spaces. Only a small number of organellar proteins have been identified in particular few proteins are known for non-luminal apicoplast compartments. We hypothesized that enlarging the catalogue of apicoplast proteins will contribute toward identifying new organellar functions and expand the realm of targets beyond a limited set of characterized pathways. We developed a bioinformatic screen based on mRNA abundance over the cell cycle and on phyletic distribution. We experimentally assessed 57 genes, and of 30 successful epitope tagged candidates eleven novel apicoplast proteins were identified. Of those, seven appear to target to the lumen of the organelle, and four localize to peripheral compartments. To address their function we then developed a robust system for the construction of conditional mutants via a promoter replacement strategy. We confirm the feasibility of this system by establishing conditional mutants for two selected genes – a luminal and a peripheral apicoplast protein. The latter is particularly intriguing as it encodes a hypothetical protein that is conserved in and unique to Apicomplexan parasites and other related organisms that maintain a red algal endosymbiont. Our studies suggest that this peripheral plastid protein, PPP1, is likely localized to the periplastid compartment. Conditional disruption of PPP1 demonstrated that it is essential for parasite survival. Phenotypic analysis of this mutant is consistent with a role of the PPP1 protein in apicoplast biogenesis, specifically in import of nuclear-encoded proteins into the organelle.