Dataset Information

Biochemical profile of the A.thaliana hypusination pathway

ABSTRACT: The eukaryotic translation elongation factor eIF5A is the only protein known to contain the unusual amino acid hypusine which is essential for its biological activity. This posttranslational modification is achieved by the sequential action of the enzymes deoxyhypusine synthase and deoxyhypusine hydroxylase. The crucial molecular function of eIF5A during translation has been recently elucidated in yeast and it is expected to be fully conserved in every eukaryotic cell, however the functional description of this pathway in plants is still scarce. The genetic approaches with transgenic plants for either eIF5A overexpression or antisense have revealed some activities related to the control of cell death processes but the molecular details remain to be characterized. One important aspect to fully understand this pathway is the biochemical description of the hypusine modification system. Here we have used recombinant eIF5A proteins either modified by hypusination or non-modified to establish a bi-dimensional electrophoresis (2D-E) profile for the three eIF5A protein isoforms present in Arabidopsis thaliana. The combined use of the recombinant 2D-E profile together with 2D-E/western blot analysis from whole plant extracts has provided a quantitative approach to measure the hypusination status of eIF5A. We have used this information to demonstrate that treatment with the hormone abscisic acid produces an alteration of the hypusine modification system in Arabidopsis thaliana. Overall this study presents the first biochemical description of the posttranslational modification of eIF5A by hypusination which will be functionally relevant for future studies related to the characterization of this pathway in Arabidopsis thaliana.

INSTRUMENT(S): 4800 Proteomics Analyzer

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

SUBMITTER: Sergio Ciordia

LAB HEAD: Dr. Alejandro Ferrando

PROVIDER: PXD000880 | Pride | 2016-07-09

REPOSITORIES: Pride

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Publications

Biochemical quantitation of the eIF5A hypusination in Arabidopsis thaliana uncovers ABA-dependent regulation.

Belda-Palazón Borja B Nohales María A MA Rambla José L JL Aceña José L JL Delgado Oscar O Fustero Santos S Martínez M Carmen MC Granell Antonio A Carbonell Juan J Ferrando Alejandro A

Frontiers in plant science 20140516

The eukaryotic translation elongation factor eIF5A is the only protein known to contain the unusual amino acid hypusine which is essential for its biological activity. This post-translational modification is achieved by the sequential action of the enzymes deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH). The crucial molecular function of eIF5A during translation has been recently elucidated in yeast and it is expected to be fully conserved in every eukaryotic cell, however the ...[more]

PMID: 24904603

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Project description:The eukaryotic translation factor eIF5A plays an essential role in translation elongation, especially across stretches of prolines and charged amino acids, and in translation termination. Although eIF5A is subjected to hypusination, a post-translational modification unique to this protein, how hypusination contributes to the eIF5A function remains elusive. Here we investigated the cellular defects induced by hypusination inhibitor GC7 (N1-guanyl-1,7-diaminoheptane). Through proteome, translatome and transcriptome analysis, we found that GC7 decreased a subset of mitochondrial proteins and DNA (mtDNA). Furthermore, chemical genomic screening using barcoded shRNA libraries identified genes for particular proteins involved in polyamine metabolism and a mitochondrial protein MPV17L2 as those altering the cytotoxicity induced by GC7. Depletion of MPV17L2 led to hypersensitivity to GC7 as well as the decreases in mitochondrial proteins and mtDNA. Moreover, metabolome analysis revealed that MPV17L2 depletion and GC7 treatment additively decreased the amount of proline and asparagine. Our results indicate that MPV17L2 displays a synthetic lethal interaction with the eIF5A hypusination targeted by GC7. The eukaryotic translation factor eIF5A plays an essential role in translation elongation, especially across stretches of prolines and charged amino acids, and in translation termination. Although eIF5A is subjected to hypusination, a post-translational modification unique to this protein, how hypusination contributes to the eIF5A function remains elusive. Here we investigated the cellular defects induced by hypusination inhibitor GC7 (N1-guanyl-1,7-diaminoheptane). Through proteome, translatome and transcriptome analysis, we found that GC7 decreased a subset of mitochondrial proteins and DNA (mtDNA). Furthermore, chemical genomic screening using barcoded shRNA libraries identified genes for particular proteins involved in polyamine metabolism and a mitochondrial protein MPV17L2 as those altering the cytotoxicity induced by GC7. Depletion of MPV17L2 led to hypersensitivity to GC7 as well as the decreases in mitochondrial proteins and mtDNA. Moreover, metabolome analysis revealed that MPV17L2 depletion and GC7 treatment additively decreased the amount of proline and asparagine. Our results indicate that MPV17L2 displays a synthetic lethal interaction with the eIF5A hypusination targeted by GC7. numerous molecules serving as valuable therapeutics. The marine natural product girolline has been described as an inhibitor of protein synthesis. Here, we demonstrate that it is not a general translation inhibitor but represents a sequence-specific modulator of translation factor eIF5A. Girolline interferes with ribosome-eIF5A interaction and induces ribosome stalling, primarily on AAA-encoded lysine. Our data furthermore indicate that eIF5A plays a physiological role in ribosome-associated quality control (RQC) and is important in maintaining the efficiency of translational progress. Girolline, therefore, provides a potent tool compound for understanding the interplay between protein production and quality control in a physiological setting and offers a new and selective means of modulating gene expression. numerous molecules serving as valuable therapeutics. The marine natural product girolline has27 been described as an inhibitor of protein synthesis. Here, we demonstrate that it is not a general28 translation inhibitor but represents a sequence-specific modulator of translation factor eIF5A.29 Girolline interferes with ribosome-eIF5A interaction and induces ribosome stalling, primarily on30 AAA-encoded lysine. Our data furthermore indicate that eIF5A plays a physiological role in31 ribosome-associated quality control (RQC) and is important in maintaining the efficiency of32 translational progress. Girolline, therefore, provides a potent tool compound for understanding33 the interplay between protein production and quality control in a physiological setting and offers34 a new and selective means of modulating gene expression.

			Action	DRS
	A1_Hyp.mgf.gz	Mgf
	A1_noHyp.mgf.gz	Mgf
	A2_Hyp.mgf.gz	Mgf
	A2_noHyp.mgf.gz	Mgf
	A3_Hyp.mgf.gz	Mgf