Dataset Information

Legionella pneumophila life stage-specific proteins regulated via ClpP-dependent

ABSTRACT: To investigate the role of ClpP-mediated proteolysis in the regulation of life stage specific proteins of Legionella pneumophila, the bacteria in the RP/TP of WT and ΔclpP were collected and analyzed by mass spectrometry. To screen the substrates of ClpP during the whole life cycle, bacterial whole-cell lysates from clpPwt and clpPtrap in the TP were prepared and His-tagged proteins were purified with Ni-NTA affinity column (GE Healthcare) following the manufacturer’s instructions. Substrates captured inside the proteolytic barrel were co-purified along with the His-tagged ClpP complex and identified by mass spectrometry to identify substrates of ClpP in the WT background. Each sample was analyzed in biological triplicates to allow for statistical tests and to improve consistency. Protein identification and quality criteria were very strict throughout the study.

ORGANISM(S): Legionella Pneumophila Subsp. Pneumophila Lp02

SUBMITTER: Yongjun Lu

PROVIDER: PXD026737 | iProX | Wed Jun 16 00:00:00 BST 2021

REPOSITORIES: iProX

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Publications

New Global Insights on the Regulation of the Biphasic Life Cycle and Virulence Via ClpP-Dependent Proteolysis in Legionella pneumophila.

Ge Zhenhuang Z Yuan Peibo P Chen Lingming L Chen Junyi J Shen Dong D She Zhigang Z Lu Yongjun Y

Molecular & cellular proteomics : MCP 20220412 5

Legionella pneumophila, an environmental bacterium that parasitizes protozoa, causes Legionnaires' disease in humans that is characterized by severe pneumonia. This bacterium adopts a distinct biphasic life cycle consisting of a nonvirulent replicative phase and a virulent transmissive phase in response to different environmental conditions. Hence, the timely and fine-tuned expression of growth and virulence factors in a life cycle-dependent manner is crucial for survival and replication. Here, ...[more]

PMID: 35427813

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Project description:We have identified loss of deiminated MA-Brent-1 (an RNA and export binding protein) in the retinal ganglion cells (RGCs) in multiple sclerosis and in glaucoma eyes compared to normal controls. Deimination refers to posttranslational modification of protein bound arginine (not free arginine) in citrulline. Our preliminary studies suggest binding of different repertoire of RNA by non-deiminated and deiminated MA-Brent-1. In vitro, in neurites of cultured RGCs and hippocampal neurons, the select mRNA translation is enhanced by addition of deiminated but not non-deiminated MA-Brent-1. These observations suggest that lack of deiminated MA-Brent-1 has consequences for protein synthesis, remodeling and plasticity of RGCs/neurons. Identification of RNA species bound by deiminated and non-deiminated MA-Brent-1 will enable us there further verification and determining the role that deimination plays in biological function of MA-Brent-1 in multiple sclerosis and glaucoma. To summarize identification of RNA species bound by deiminated and non deiminated MA-Brent-1 will enable us to gain further insight into role of deimination in the overall disease process. To identify the RNA species bound by deiminated and non-deiminated MA-Brent-1 The deiminanted MA-Brent-1 binds to a different repertoire of RNA and non-deiminated MA-Brent-1. Corollary to this hypothesis is that the deiminated MA-Brent-1 enhances translation for bound species of RNA. Loss of deimination results in less dendritic protein synthesis and has consequences for remodeling and plasticity in multiple sclerosis and glaucoma RGCs where lack of deimination (compared to control eyes) has been observed. About 10 microgram of purified recombinant and his-tagged non-deiminated MA-Brent-1 or deiminated MA-Brent-1 will be incubated with about 400 microgram of total RNA. The RNA bound his-tagged protein will be incubated with about 100 microliters of Ni-NTA beads and loaded onto a mini column. The column will be washed with 50 volumes of binding buffer. The bound his-tagged protein will be released using 100 mM immidazole in the binding buffer. The MA-Brent-1 bound RNA will be recovered by boiling the protein in 0.25% SDS followed by purification of RNA using columns or using a 4% PAGE-0.8% agarose composite gel followed by gel purification. The composite gel RNA purification is routine practice in the laboratory. RNA binding experiment for each protein variant (deiminated or non-deiminated) MA-Brent-1 will be repeated twice. Identified RNA species common in both binding experiments for a given species will be candidates for further pursuit. The recombinant purified MA-Brent-1 will be subjected to deimination by incubating with recombinant peptidylarginine deiminase type II (PAD2). PAD2 is a C-terminus GST tag fusion protein and will be purified using GST column. Deiminated and non-deiminated MA-Brent-1, C-terminal his-tag fusion proteins will be purified using Ni-NTA beads/column and immidazole containing buffer as eluents.

Project description:We have identified loss of deiminated MA-Brent-1 (an RNA and export binding protein) in the retinal ganglion cells (RGCs) in multiple sclerosis and in glaucoma eyes compared to normal controls. Deimination refers to posttranslational modification of protein bound arginine (not free arginine) in citrulline. Our preliminary studies suggest binding of different repertoire of RNA by non-deiminated and deiminated MA-Brent-1. In vitro, in neurites of cultured RGCs and hippocampal neurons, the select mRNA translation is enhanced by addition of deiminated but not non-deiminated MA-Brent-1. These observations suggest that lack of deiminated MA-Brent-1 has consequences for protein synthesis, remodeling and plasticity of RGCs/neurons. Identification of RNA species bound by deiminated and non-deiminated MA-Brent-1 will enable us there further verification and determining the role that deimination plays in biological function of MA-Brent-1 in multiple sclerosis and glaucoma. To summarize identification of RNA species bound by deiminated and non deiminated MA-Brent-1 will enable us to gain further insight into role of deimination in the overall disease process. To identify the RNA species bound by deiminated and non-deiminated MA-Brent-1; The deiminanted MA-Brent-1 binds to a different repertoire of RNA and non-deiminated MA-Brent-1. Corollary to this hypothesis is that the deiminated MA-Brent-1 enhances translation for bound species of RNA. Loss of deimination results in less dendritic protein synthesis and has consequences for remodeling and plasticity in multiple sclerosis and glaucoma RGCs where lack of deimination (compared to control eyes) has been observed. About 10 microgram of purified recombinant and his-tagged non-deiminated MA-Brent-1 or deiminated MA-Brent-1 will be incubated with about 400 microgram of total RNA. The RNA bound his-tagged protein will be incubated with about 100 microliters of Ni-NTA beads and loaded onto a mini column. The column will be washed with 50 volumes of binding buffer. The bound his-tagged protein will be released using 100 mM immidazole in the binding buffer. The MA-Brent-1 bound RNA will be recovered by boiling the protein in 0.25% SDS followed by purification of RNA using columns or using a 4% PAGE-0.8% agarose composite gel followed by gel purification. The composite gel RNA purification is routine practice in the laboratory. RNA binding experiment for each protein variant (deiminated or non-deiminated) MA-Brent-1 will be repeated twice. Identified RNA species common in both binding experiments for a given species will be candidates for further pursuit. The recombinant purified MA-Brent-1 will be subjected to deimination by incubating with recombinant peptidylarginine deiminase type II (PAD2). PAD2 is a C-terminus GST tag fusion protein and will be purified using GST column. Deiminated and non-deiminated MA-Brent-1, C-terminal his-tag fusion proteins will be purified using Ni-NTA beads/column and immidazole containing buffer as eluents. Experiment Overall Design: in vitro RNA binding with deiminated and non-deiminated MA-Brent-1

Dataset Information

Legionella pneumophila life stage-specific proteins regulated via ClpP-dependent

Publications

New Global Insights on the Regulation of the Biphasic Life Cycle and Virulence Via ClpP-Dependent Proteolysis in Legionella pneumophila.

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