Dataset Information

SILAC Phospho-proteomics: Trypanosoma brucei

ABSTRACT: Pseudokinases can modulate activity of protein kinases, among other modes of influencing biological pathways. We tested a hypothesis that knockdown of Tb427tmp.160.4770 (a Pseudokinase) perturbs the Phospho-proteome of T. brucei. To discover changes in the trypanosome phospho-proteome, stable isotope labelling of amino acids in cell culture (SILAC) was performed, followed by enrichment of phosphopeptides with immobilised metal affinity chromatography (IMAC), and phospho-peptide detection/quantitation using mass spectroscopy. Trypanosomes (p2T7-Tb4770) were cultured in heavy (13C6-L-Arginine, 2H4-L-Lysine) or light isotopes (L-Arginine, L-Lysine) for SILAC. Trypanosomes grown in heavy medium were induced with tetracycline for knockdown of Tb427tmp.160.4770. Induced (Tet+, H) and uninduced (Tet-, L) samples were combined and processed together for IMAC and mass spectroscopy

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Trypanosoma Brucei

DISEASE(S): Trypanosomiasis

SUBMITTER: Gaurav Kumar

LAB HEAD: Kojo-Mensa Wilmot

PROVIDER: PXD034516 | Pride | 2023-03-11

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	BT062117_071217_AAK1_tet_minus_vs_tet_minus_6_19_IMAC_EL_0.msf	Msf
	BT062117_071217_AAK1_tet_minus_vs_tet_minus_6_19_IMAC_EL_01.raw	Raw
	BT062117_071217_AAK1_tet_minus_vs_tet_minus_6_19_IMAC_EL_02.raw	Raw
	BT062117_071217_AAK1_tet_minus_vs_tet_plus_5_29_IMAC_EL_0.msf	Msf
	BT062117_071217_AAK1_tet_minus_vs_tet_plus_5_29_IMAC_EL_01.raw	Raw

Items per page:

1 - 5 of 13

Publications

Pseudokinase NRP1 facilitates endocytosis of transferrin in the African trypanosome.

Kumar Gaurav G Thomas Bryanna B Mensa-Wilmot Kojo K

Scientific reports 20221103 1

Trypanosoma brucei causes human African trypanosomiasis (HAT) and nagana in cattle. During infection of a vertebrate, endocytosis of host transferrin (Tf) is important for viability of the parasite. The majority of proteins involved in trypanosome endocytosis of Tf are unknown. Here we identify pseudokinase NRP1 (Tb427tmp.160.4770) as a regulator of Tf endocytosis. Genetic knockdown of NRP1 inhibited endocytosis of Tf without blocking uptake of bovine serum albumin. Binding of Tf to the flagella ...[more]

PMID: 36329148

Similar Datasets

Project description:RPF-1, a transcription factors encoded by POU6F2 gene, appears to be expressed in the developing nervous system and embryonic kidney. We generated a stable HEK/RPF-1 transfectant expressing the protein in a tetracycline (Tet)-dependent induction and addressed the whole transcriptome regulated by RPF-1. We detected a gross alteration of gene expression that is consistent with its occupancy at proximal promoters, and gel retardation assay and promoter-reporter activity, addressing a few target genes, indicated a dependence by RPF-1 of transcriptional response. GO analysis supports a role for this factor in the genetic programs guiding early differentiation of embryonic kidney and neuronal fate. RPF-1, a transcription factors encoded by POU6F2 gene, appears to be expressed in the developing nervous system and embryonic kidney. We generated a stable HEK/RPF-1 transfectant expressing the protein in a tetracycline (Tet)-dependent induction and addressed the whole transcriptome regulated by RPF-1. We detected a gross alteration of gene expression that is consistent with its occupancy at proximal promoters, and gel retardation assay and promoter-reporter activity, addressing a few target genes, indicated a dependence by RPF-1 of transcriptional response. GO analysis supports a role for this factor in the genetic programs guiding early differentiation of embryonic kidney and neuronal fate. Gene array analysis shows that RPF-1 transcription factor modify the transcriptome of HEK293 cells by regulating a genetic program endowed with developmental programs Comparison of HEK293 cells induced for RPF-1 expression (Tet-) over uninduced control (Tet+) and Mock cells. RPF-1 cDNA was inserted into the pTRE2puro vector to generate stable Tet-inducible KEK293 transfectants. Upon Tet removal from the medium, HEK/RPF-1 transfectants expressed the transcription factor RPF-1. Cells were then seeded in 10 cm dishes, cultured for 48 h either in complete medium supplemented with Tet (+) or in Tet-depleted medium (-), harvested, and spun down before RNA isolation. Comparative analysis between HEK/RPF-1 induced (Tet-) and uninduced (Tet+) transfectants allowed us to identify genes transcriptionally regulated by RPF-1. Residual effects on gene expression due to Tet supplementation into culture media was ruled out by comparison with the Mock transfectant.

Project description:The host range of African trypanosomes is influenced by innate protective molecules in the blood of primates. A subfraction of human high-density lipoprotein (HDL) containing apolipoprotein A-I, apolipoprotein L-I, and haptoglobin-related protein is toxic to Trypanosoma brucei brucei but not the human sleeping sickness parasite Trypanosoma brucei rhodesiense. It is thought that T. b. rhodesiense evolved from a T. b. brucei-like ancestor and expresses a defense protein that ablates the antitrypanosomal activity of human HDL. To directly investigate this possibility, we developed an in vitro selection to generate human HDL-resistant T. b. brucei. Here we show that conversion of T. b. brucei from human HDL sensitive to resistant correlates with changes in the expression of the variant surface glycoprotein (VSG) and abolished uptake of the cytotoxic human HDLs. Complete transcriptome analysis of the HDL-susceptible and -resistant trypanosomes confirmed that VSG switching had occurred but failed to reveal the expression of other genes specifically associated with human HDL resistance, including the serum resistance-associated gene (SRA) of T. b. rhodesiense. In addition, we found that while the original active expression site was still utilized, expression of three expression site-associated genes (ESAG) was altered in the HDL-resistant trypanosomes. These findings demonstrate that resistance to human HDLs can be acquired by T. b. brucei. Keywords: Trypanosoma, VSG, antigenic switching, HDL-resistance Bloodstream stages of the Lister strain 427 T. b. brucei (MiTat 1.2), expressing VSG221, were used in these studies. Cells were cultured in HMI-9 medium with the addition of heat inactivated fetal bovine serum (FBS) (10%) and Serum Plus (10%). T. b. brucei 427-221 is an antigenically stable line and contains a single copy of the vsg221 gene within the 221 expression site (221ES). At a cell density of approximately 1,000,000 cells/ml, T. b. brucei 427-221 were exposed to various amounts of human HDLs for 24 h in a 6 well plate. Surviving trypanosomes were counted using a hemocytometer then diluted into fresh HMI-9 medium and allowed to recover for 5-14 days. Once the cells had grown to a density of approximately 1,000,000 cells/ml, they were once again incubated with human HDLs. Each round of selection was performed with increasing concentrations of human HDLs and freezer stocks were prepared for each surviving population. Over nine months we conducted eight rounds of human HDL selection, resulting in a population of T. b. brucei that survived incubation with 800 μl of human HDLs (160 lytic U).