Phosphoproteomic studies to identify targets and substrates of Protein kinase PfPK2 in human malaria parasite Plasmodium falciparum
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Fusion
ORGANISM(S): Plasmodium Falciparum (isolate 3d7)
SUBMITTER: 
Keshava Prasad T. S.
LAB HEAD: T. S. Keshava Prasad, Ph.D.
PROVIDER: PXD042264 | Pride | 2023-10-18
REPOSITORIES: Pride
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| IGSDC_P_F1_R1.raw | Raw | |||
| IGSDC_P_F1_R2.raw | Raw | |||
| IGSDC_P_F2_R1.raw | Raw | |||
| IGSDC_P_F2_R2.raw | Raw | |||
| IGSDC_P_F3_R1.raw | Raw |
Items per page: 5 1 - 5 of 58 |
Similar Datasets
Project description:Calcium Dependent Protein Kinases (CDPKs) play important function in calcium signaling at various stage of Plasmodium falciparum. CDPK7 is one such kinase, which plays a crucial role in the development of Plasmodium falciparum parasite. In order to understand the function of PfCDPK7, we performed comparative phosphoproteomic analysis of PfCDPK7 knockout mutant and wild-type lines. In brief, proteins from PfCDPK7 knockout mutant and wild-type parasites were isolated, digested with trypsin and labelled with iTRAQ tags. Phosphopeptides were then enriched from iTRAQ labeled peptides and subjected to LC-MS/MS analysis. This led to the identification of putative substrates of PfCDPK7 in the parasites. A comparative proteomic analysis was performed to measure the protein abundance changes in the PfCDPK7 mutant parasites.
2021-11-08 | PXD025384 | Pride
Project description:Calcium Dependent Protein Kinases (CDPKs) are key effector in calcium signaling in Apicomplexan parasites. CDPK7 is one such kinase, which plays a crucial role in intracellular infection of Toxoplasma gondii parasite. To gain insights into how CDPK7 play a role in parasites survival, we have performed comparative phosphoproteomic to identify putative targets of TgCDPK7 in the parasites. Proteins from TgCDPK7 knockdown and wild were isolated, digested with trypsin and labelled with TMT. From the quantitative proteomic analysis by high- resolution mass spectrometry, differentially phosphorylated proteins were identified, which can be direct or indirect substrates of TgCDPK7. Many proteins which are involved in lipid metabolism are also found significantly altered.
2021-04-27 | PXD019655 | Pride
Project description:Calcium Dependent Protein Kinases (CDPKs) are key effectors of calcium signaling in malaria parasite. We have elucidated a novel role of PfCDPK1, which is indispensable for the erythrocytic development of Plasmodium falciparum, in invasion of host Red Blood Cells (RBCs). To gain insights into the mechanisms via which PfCDPK1 is involved in parasitic processes, a comparative phosphoproteomic analysis was employed to identify its parasitic targets. Proteins were isolated from CDPK1 knockdown and wild type parasites and processed for trypsin digestion and iTRAQ labeling. Quantitative proteomic analysis using high resolution mass spectrometry led to the identification of hypophosphorylated proteins, which can be the likely substrates for this kinase. In addition, in vitro kinase assays and antibody based validation of the hypophosphorylated sites was carried out. This study will provide a novel insight into the CDPK1 mediated invasion by Plasmodium falciparum.
2017-05-02 | PXD005207 | Pride
Project description:The P. falciparum genome is equipped with several subtelomeric gene families that are implicated in parasite virulence and immune evasion. The members of these gene families are uniformly positioned within heterochromatic domains of the genome and are thus subject to variegated expression. The best-studied example is that of the var gene family encoding the major parasite virulence factor P. falciparum erythrocyte membrane protein 1 (PfEMP1). Transcriptional regulation of other subtelomeric gene families and their role in parasite biology is much less understood. Here, we investigated the mode of transcriptional control of var, rif, stevor, phist and pfmc-2tm families by comparative genome-wide transcriptional profiling of transgenic parasite lines. Our results establish a clear functional distinction between var and non-var transcriptional control mechanisms. Unlike var promoters, we find that promoters of non-var families are not silenced by default. Moreover, we show that mutually exclusive transcription is unique to the var gene family. 3D7 wild-type parasites were transfected with constructs carrying eight different promoters that drive expression of the drug-selectable marker hdhfr-gfp. Thereof seven promoters are members of the multigene families upsA var, upsB var, upsC var, rif, stevor, phistb and pfmc-2tm. The cam promoter was used as transfection-based control and also a wild-type 3D7 cell line was included as control. These nine cell lines were subjected to genome-wide transcriptional profiling. Parasites were synchronized to obtain an 8 hour growth window and were harvested at four consecutive timepoints (TP): TP1 (6-14 hours post-invasion (hpi)); TP2 (14-22 hpi); TP3 (22-30 hpi); TP4 (30-38 hpi) to monitor intra- and inter-family specific linkage of multigene family expression.
2011-09-01 | E-GEOD-31829 | biostudies-arrayexpress
Project description:Plasmodium yoelii YM asexual blood stage parasites express multiple members of the py235 gene family, part of the super-family of genes including those coding for Plasmodium vivax reticulocyte binding proteins and Plasmodium falciparum RH proteins. Dr Tony Holder's laboratory (NIMR, London) has been successful in deleting one of the RH family genes (Py01365) by transfection and insertion of the TgDHFR gene, and cloned the resulting parasite in YM background. The gene expression patterns of the mutant parasite line were compared to that of the wild type YM parasite.
2011-04-25 | E-MTAB-636 | biostudies-arrayexpress
Project description:To address whether T. brucei has a circadian clock we probed its transcriptome by RNA-seq, searching for transcripts oscillating with a 24 hr period. For this we entrained/ synchronized parasites in vitro for three days using temperature and light as environmental stimuli and then collected parasite RNA every four hours for two consecutive days. Parasite RNA was subjected to RNA-seq analysis. We performed this protocol on two stages of the T. brucei life cycle (bloodstream and insect procyclic forms).
2017-02-10 | E-MTAB-4952 | biostudies-arrayexpress
Project description:We described the biological role of a putative zinc finger protein (Plasmodb id: PF3D7_1008600), which plays an important role during the initiation and subsequent progression of gametocytogenesis in P. falciparum. Hence we termed the protein P. falciparum Zinc Finger Protein for Gametocytogenesis (PfZnFP-G). During the asexual intraerythrocyic developmental cycle (IDC), PfZnFP-G levels peak during the schizont stage during which it is localized in the nucleus. During the IDC, PfZnFP-G appears to be stochastically expressed in ~3% parasite population. Deactivation of PfZnFP-G appears to suppress, but not fully inhibit, production of gametocytes and vice versa overproduction of PfZnFP-G stimulates gametocytogenesis. In addition, PfZnFP-G is capable of associating with DNA binding preferentially to intergenic, promoter regions of the genome, and this binding correlates positively with transcription, particularly of other gametocyte specific genes. PfZnFP-G is also expressed in the mid-to-late gametocytes where it is present in the cytoplasm. Overall, these results suggest a dual function of PfZnFP-G in gametocytogenesis including: (i) commitment as a transcription factor and (ii) gametocyte maturation as a putative RNA binding protein. Transgenic 3D7 wild type parasite lines were generated with PfZnFP-G tagged, over-expressed and knocked-out. Transcriptional profiles across the parasite life cycle were generated. Genome occupancy of PfZnFP-G were also investigated and compared to the corresponding transcriptional profiles.
2015-11-24 | E-GEOD-75295 | biostudies-arrayexpress
Project description:To study the bromodomain protein PfBDP1 in the malaria parasite P. falciparum, a transgenic parasite line was generated (PfBDP1DD) in which PfBDP1 was tagged with a haemagglutinin tag and a ligand regulatable FKBP destabilization domain that allowed conditional knockdown of PfBDP1 by removal of the stabilizing ligand Shld1 from cultures. Shld1 was removed 30 hours post invasion (hpi) and the cells allowed to reinvade fresh red blood cells. The effects of PfBDP1 knockdown on gene expression were assessed by transcriptional profiling on microarrays over 6 consecutive time points (8 hour intervals) in the intraerythrocytic developmental cycle (IDC). Two condition matched time course experiment, PfBDP1DD ON versus OFF Shld1. Transgenic P. falciparum 3D7 parasites (PfBDP1DD) expressing an endogeneous PfBDP1-HA-DD fusion protein were grown in the presence of 2.5 nM WR99210/500 nM Shld1 (PfBDP1DD_ON) or 2.5 nM WR99210 (PfBDP1DD_OFF). After invasion, RNA was extracted at 6 consectutive time points in 8 hour intervals during the IDC, starting at 4 hpi, and was processed for microarray analysis. Three matched biological replicates were performed for both conditions, independently grown and harvested. Each array represents one RNA sample.
2015-01-07 | E-GEOD-64688 | biostudies-arrayexpress
Project description:Malaria infection triggers vigorous host immune responses; however, the parasite ligands, host receptors and the signaling pathways responsible for these reactions remain unknown or controversial. Malaria parasites primarily reside within red blood cells (RBCs), thereby hiding themselves from direct contact and recognition by host immune cells. Host responses to malaria infection are very different from those elicited by bacterial and viral infections and the host receptors recognizing parasite ligands have been elusive. Here we investigated mouse genome-wide transcriptional responses to infections with two strains of Plasmodium yoelii (N67 and N67C) and discovered differences in innate response pathways corresponding to strain-specific disease phenotypes. Using in vitro RNAi gene knockdown and knockout mice, we demonstrated that a strong IFN-I response triggered by RNA Polymerase III and melanoma differentiation-associated protein 5 (MDA5), not Toll-like receptors (TLRs), binding of parasite DNA/RNA contributed to a decline of parasitemia in N67-infected mice. We showed that conventional dendritic cells were the major sources of early IFN-I, and that surface expression of phosphatidylserine (PS) on infected RBC (iRBC) might promote their phagocytic uptake, leading to the release of parasite ligands and the IFN-I response in N67 infection. In contrast, an elevated inflammatory response mediated by CD14/TLR and p38 signaling played a role in disease severity and early host death in N67C-infected mice. In addition to identifying cytosolic DNA/RNA sensors and signaling pathways previously unrecognized in malaria infection, our study demonstrates the importance of parasite genetic backgrounds in malaria pathology and provides important information for studying human malaria pathogenesis. Spleen RNA from mice, 4 days post infection with Plasmodium yoelii (strain N67 or N67C), or mock infection (N). Replicates from 6 individual mice per condition.
2014-01-13 | E-GEOD-51329 | biostudies-arrayexpress
Project description:Quantitative studies of the P. falciparum transcriptome have shown that the tightly controlled progression of the parasite through the intraerythrocytic developmental cycle (IDC) is accompanied by a continuous gene expression cascade where most expressed genes exhibit a single transcriptional peak. Since proteins represent the decisive business end of gene expression, understanding the correlation between mRNA and protein levels is crucial for inferring biological activity from transcriptional gene expression data. While pertinent studies on other organisms show that as little as 20-40% of protein abundance variation may be attributable to corresponding mRNA levels, the situation in Plasmodium is further complicated by the dynamic nature of the cyclic gene expression cascade where the mRNA levels of most genes change constantly during the IDC. In this study, we simultaneously determined mRNA and protein abundance profiles for P. falciparum parasites during the IDC at 2-hour resolution based on spotted oligonucleotide microarrays and 2D-protein gels in combination with DIGE fluorescent dyes. Intriguingly, most proteins are represented by more than one isoform, presumably due to post-translational modifications. Analysis of 366 protein abundance profiles and the corresponding mRNA levels shows that in 67.2% of cases the protein abundance peaks at least 8 hours after the mRNA level peak. While it may be tempting to interpret this as evidence for widespread post-transcriptional gene regulation additional analyses including computer modeling demonstrate that in >60% of these cases the observed protein profiles including the peak lag times could arise as a consequence of the corresponding mRNA levels when simple translation and degradation dynamics are assumed. We further characterize and illustrate these dynamics and show that even human host proteins within the parasite may be subject to similar dynamics as their parasite counterparts. 24 timepoint samples were harvested from a tightly synchronous 6.5 liter biofermenter culture of P. falciparum (Dd2) at 2-hour intervals during one entire intraerythrocytic developmental cycle and compared against a 3D7 RNA reference pool.
2011-06-07 | E-GEOD-24416 | biostudies-arrayexpress