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PfDOZI protein-protein interaction in plasmodium falciparum

ABSTRACT: Here, we characterized an RNA binding protein (RBP), PfDOZI, which is an integral cytoplasmic component of RNP granule. We observed that deleting pfdozi led to an asexual growth advantage due to the escalated merozoite invasion capacity. Also, pfdozi disruption resulted in a significant impairment in gametocyte commitment and morphology development. Through high-throughput RNA-seq, we revealed that PfDOZI affected the transcriptome in both asexual and sexual stages when pfdozi was deleted. Mechanically, we confirmed that PfDOZI could interact with P body like mRNP in the schizont stage, while stress granule-like granule in gametocyte stage. The identification of PfDOZI protein complex in stressed schizont further indicate PfDOZI could shift its function from mRNA degradation to translation repression during stage conversion.

INSTRUMENT(S): orbitrap

ORGANISM(S): Plasmodium Falciparum

TISSUE(S): Life Cycle Stage

DISEASE(S): Plasmodium Falciparum Malaria

SUBMITTER: HUI MIN

LAB HEAD: Liwang Cui

PROVIDER: PXD020862 | Pride | 2024-04-16

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	20190807_MinHui-1_Doz-GFP_Schi.raw	Raw
	20190807_MinHui-1_Doz-GFP_Schi__F157477_.mzid.gz	Mzid
	20190807_MinHui-1_Doz-GFP_Schi__F157477_.mzid_20190807_MinHui-1_Doz-GFP_Schi__F157477_.MGF	Mzid
	20190807_MinHui-4_Doz-GFP_GAD3-Control.raw	Raw
	20190807_MinHui-4_Doz-GFP_GAD3-Control__F157480_.mzid.gz	Mzid

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Project description:During intra-erythrocytic development, late asexually replicating Plasmodium falciparum parasites sequester from peripheral circulation. This facilitates chronic infection and is linked to severe disease and organ-specific pathology including cerebral and placental malaria. Immature gametocytes M-bM-^@M-^S sexual stage precursor cells M-bM-^@M-^S likewise disappear from circulation. Recent work has demonstrated that these sexual stage parasites are located in the hematopoietic system of the bone marrow before mature gametocytes are released into the blood stream to facilitate mosquito transmission. However, as sequestration occurs only in vivo and not during in vitro culture, the mechanisms by which it is regulated and enacted (particularly by the gametocyte stage) remain poorly understood. We generated the most comprehensive P. falciparum functional gene network to date by integrating global transcriptional data from a large set of asexual and sexual in vitro samples, patient-derived in vivo samples, and a new set of in vitro samples profiling sexual commitment. We defined more than 250 functional modules (clusters) of genes that are co-expressed primarily during the intra-erythrocytic parasite cycle, including 35 during sexual commitment and gametocyte development. Comparing the in vivo and in vitro datasets allowed us, for the first time, to map the time point of asexual parasite sequestration in patients to 22 hours post invasion, confirming previous in vitro observations on the dynamics of host cell modification and cytoadherence. Moreover, we were able to define the properties of gametocyte sequestration, demonstrating the presence of two circulating gametocyte populations: gametocyte rings between 0 and ~30 hours post invasion and mature gametocytes after around 7 days post invasion. We used 164/TdTom, a transgenic parasite line expressing a red fluorescent protein reporter under a gametocyte-specific promoter to generate schizont samples. Schizonts were subsequently isolated from both the fluorescent and non-fluorescent population by FACS and prepared for microarray analysis. Two biological replicates were produced for both the fluorescent and the non-fluorescent samples.

Project description:Ubiquitylation is a common post translational modification of eukaryotic proteins. Protein ubiquitylation increases in the transition from intracellular schizont to extracellular merozoite in the asexual blood stage cycle of the human malaria parasite, Plasmodium falciparum (Pf). Here, we identify specific ubiquitylation sites of protein substrates in three intracellular parasite stages and extracellular merozoites; a total of 1464 sites in 546 proteins were identified. 469 ubiquitylated proteins were identified in merozoites compared with only 160 in the preceding intracellular schizont stage, indicating a large increase in protein ubiquitylation associated with merozoite maturation. Following merozoite invasion of erythrocytes, few ubiquitylated proteins were detected in the first intracellular ring stage but as parasites matured through trophozoite to schizont stages the extent of ubiquitylation increased. We identified commonly used ubiquitylation motifs and groups of ubiquitylated proteins in specific areas of cellular function, for example merozoite pellicle proteins involved in erythrocyte invasion, exported proteins, and histones. To investigate the importance of ubiquitylation we screened ubiquitin pathway inhibitors in a parasite growth assay and identified the ubiquitin activating enzyme (UBA1 or E1) inhibitor, MLN7243 (TAK-243) to be particularly effective. This small molecule was shown to be a potent inhibitor of recombinant PfUBA1, and a structural homology model of MLN7243 bound to the parasite enzyme highlights avenues for the development of P. falciparum specific inhibitors. We constructed a genetically modified parasite with a rapamycin-inducible functional deletion of uba1; addition of either MLN7243 or rapamycin to the recombinant parasite line resulted in the same phenotype, with parasite development blocked at the schizont stage. These results indicate that the intracellular target of MLN7243 is UBA1, and this activity is essential for the final differentiation of schizonts to merozoites. The ubiquitylation of many merozoite proteins and their disappearance in ring stages are consistent with the idea that ubiquitylation leads to their destruction via the proteasome once their function is complete following invasion, which would allow amino acid recycling in the period prior to the parasite’s elaboration of a new food vacuole.

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PfDOZI protein-protein interaction in plasmodium falciparum

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