Dataset Information

Label-free, quantitative analysis of PKA-dependent phosphoproteome changes in Trypanosoma brucei

ABSTRACT: Protein kinase A (PKA), the main effector of second messenger cAMP, is highly conserved among eukaryotes and a paradigm for the mechanisms of regulation of protein kinases by ligands. The unique PKA holoenzymes in the phylogenetically distant protozoan parasite Trypanosoma are unresponsive to cAMP in vitro and in vivo. By small molecule screening and optimization, we designed direct, membrane-permeable activators binding with a kD of 9 nM to the CNB pockets of the T. brucei regulatory PKA subunit. 7-Cyano-7-deazainosine has low toxicity and thus is a perfect tool to explore cAMP-independent PKA signaling in these important pathogens. This project describes PKA-induced phosphoproteome changes of the bloodstream stage of T. brucei.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Trypanosoma Brucei

SUBMITTER: Ignasi Forne

LAB HEAD: Prof. Dr. Michael Boshart (Genetics, Biochemistry, Cell Biology) Prof. Dr. Michael Boshart

PROVIDER: PXD012245 | Pride | 2019-02-25

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	Ref1976_MB_S01_Ph_20181128.raw	Raw
	Ref1976_MB_S01_Pr_20181128.raw	Raw
	Ref1976_MB_S02_Ph_20181128.raw	Raw
	Ref1976_MB_S02_Pr_20181128.raw	Raw
	Ref1976_MB_S03_Ph_20181128.raw	Raw

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Publications

Nucleoside analogue activators of cyclic AMP-independent protein kinase A of Trypanosoma.

Bachmaier Sabine S Volpato Santos Yuri Y Kramer Susanne S Githure George Boniface GB Klöckner Thomas T Pepperl Julia J Baums Cordula C Schenk Robin R Schwede Frank F Genieser Hans-Gottfried HG Dupuy Jean-William JW Forné Ignasi I Imhof Axel A Basquin Jerôme J Lorentzen Esben E Boshart Michael M

Nature communications 20190329 1

Protein kinase A (PKA), the main effector of cAMP in eukaryotes, is a paradigm for the mechanisms of ligand-dependent and allosteric regulation in signalling. Here we report the orthologous but cAMP-independent PKA of the protozoan Trypanosoma and identify 7-deaza-nucleosides as potent activators (EC<sub>50</sub> ≥ 6.5 nM) and high affinity ligands (K<sub>D</sub> ≥ 8 nM). A co-crystal structure of trypanosome PKA with 7-cyano-7-deazainosine and molecular docking show how substitution of key amin ...[more]

PMID: 30926779

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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).