Interaction of 2',3'-cAMP with Rbp47b plays a role in stress granule formation

Kosmacz, Monika; Luzarowski, Marcin; Kerber, Olga; Leniak, Ewa; Gutiérrez-Beltrán, Emilio; Moreno, Juan C.; Górka, Michał; Szlachetko, Jagoda; Veyel, Daniel; Graf, Alexander

doi:10.1104/pp.18.00285

Cited by 39 publications

(62 citation statements)

References 49 publications

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“…Obtained results demonstrate that increase in the 2 0 ,3 0 -cAMP level is not obligatory for SGs formation. Rather, as speculated previously, 2 0 ,3 0 -cAMP facilitates SGs formation under conditions associated with an excessive RNA degradation (Kosmacz et al, 2018). Phospholipids also showed differential responses, which resulted in a coordinated decrease in phospholipids levels in the WT but not in the 35S:GFP-Rbp47 seedlings (Fig.…”

Section: Metabolome Composition Of the Arabidopsis Sgssupporting

confidence: 67%

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Protein and metabolite composition of Arabidopsis stress granules

et al. 2019

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Summary Stress granules (SGs) are evolutionary conserved aggregates of proteins and untranslated mRNAs formed in response to stress. Despite their importance for stress adaptation, no complete proteome composition has been reported for plant SGs. In this study, we addressed the existing gap. Importantly, we also provide evidence for metabolite sequestration within the SGs. To isolate SGs we used Arabidopsis seedlings expressing green fluorescent protein (GFP) fusion of the SGs marker protein, Rbp47b, and an experimental protocol combining differential centrifugation with affinity purification (AP). SGs isolates were analysed using mass spectrometry‐based proteomics and metabolomics. A quarter of the identified proteins constituted known or predicted SG components. Intriguingly, the remaining proteins were enriched in key enzymes and regulators, such as cyclin‐dependent kinase A (CDKA), that mediate plant responses to stress. In addition to proteins, nucleotides, amino acids and phospholipids also accumulated in SGs. Taken together, our results indicated the presence of a preexisting SG protein interaction network; an evolutionary conservation of the proteins involved in SG assembly and dynamics; an important role for SGs in moderation of stress responses by selective storage of proteins and metabolites.

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Section: Metabolome Composition Of the Arabidopsis Sgssupporting

confidence: 67%

“…Obtained results demonstrate that increase in the 2′,3′‐cAMP level is not obligatory for SGs formation. Rather, as speculated previously, 2′,3′‐cAMP facilitates SGs formation under conditions associated with an excessive RNA degradation (Kosmacz et al ., ).…”

Section: Resultsmentioning

confidence: 97%

Protein and metabolite composition of Arabidopsis stress granules

et al. 2019

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“…Extracellular 2′,3′-cAMP is metabolized to 2′-AMP and 3′-AMP, which in turn are metabolized to adenosine 14 . Importantly, intracellular 2′,3′-cAMP opens mitochondrial permeability transition pores (mPTPs 15 ); and triggers stress granule formation 16 . Thus extracellular 2′,3′-cAMP can engage A 2B R via adenosine, and intracellular 2′,3′-cAMP can compromise cellular energy production via opening mPTPs and stimulating the production of stress granules.…”

Section: -Br-2′3′-camp Enhances Exosome Production and The Effects mentioning

confidence: 99%

Simultaneous Inhibition of Glycolysis and Oxidative Phosphorylation Triggers a Multi-Fold Increase in Secretion of Exosomes: Possible Role of 2′,3′-cAMP

Ludwig

Yerneni

Menshikova

et al. 2020

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exosome secretion by cells is a complex, poorly understood process. Studies of exosomes would be facilitated by a method for increasing their production and release. Here, we present a method for stimulating the secretion of exosomes. cultured cells were treated or not with sodium iodoacetate (IAA; glycolysis inhibitor) plus 2,4-dinitrophenol (DNP; oxidative phosphorylation inhibitor). Exosomes were isolated by size-exclusion chromatography and their morphology, size, concentration, cargo components and functional activity were compared. IAA/DNP treatment (up to 10 µM each) was nontoxic and resulted in a 3 to 16-fold increase in exosome secretion. Exosomes from IAA/DNP-treated or untreated cells had similar biological properties and functional effects on endothelial cells (SVEC4-10). iAA/Dnp increased exosome secretion from mouse organ cultures, and in vivo injections enhanced the levels of circulating exosomes. iAA/Dnp decreased Atp levels (p < 0.05) in cells. A cell membranepermeable form of 2′,3′-cAMP and 3′-AMP mimicked the potentiating effects of IAA/DNP on exosome secretion. In cells lacking 2′,3′-cyclic nucleotide 3′-phosphodiesterase (cnpase; an enzyme that metabolizes 2′,3′-cAMP into 2′-AMP), effects of IAA/DNP on exosome secretion were enhanced. The IAA/DNP combination is a powerful stimulator of exosome secretion, and these stimulatory effects are, in part, mediated by intracellular 2′,3′-cAMp. Extracellular vesicles (EVs), including the small subset of EVs referred to as exosomes which are derived from the endocytic compartment of parental cells and range in size from 30 to 150 nm, play an important role in intercellular communication. The biogenesis of exosomes is distinct from that of other EVs, such as microvesicles (MVs) or apoptotic bodies 1. It begins with the internalization of cell surface proteins by endocytosis and the sequestration of these proteins by early endosomes. In late endosomes, a process of reverse vesicular invagination leads to the formation of multivesicular bodies (MVBs), which are filled with numerous vesicles. Importantly the topography of proteins decorating these vesicles mimics that of the surface membrane of a parental cell 2. Thus, exosomes differ from other EVs in that their vesicular cargo is derived from the proteins processed in late endosomes and packaged into vesicles in the MVBs. When MVBs fuse with the cell membrane of the parent cell, exosomes are released into the extracellular space 3. Exosome packaging and their cellular secretion have been extensively investigated and while the general mechanistic underpinning their formation are described, it remains unclear to which extent the packaged and secreted exosomes are molecular mimics of their parental cells or whether they

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“…Alternative strategies rely on biochemical methods and can be divided into three groups: (i) ligand‐targeted, in which a metabolite is used as bait to determine the targets of the small molecule; (ii) protein‐targeted, which aim to identify small molecules affecting the function of a signaling protein; and (iii) untargeted, which chart a map of the protein–metabolite interactome in a given organism (Luzarowski & Skirycz, ). Examples of ligand‐targeted approaches include classical strategies like affinity purification, in which a chemical compound is immobilized to the matrix and used to capture interacting proteins from native cellular lysate, as well as the recent methods TPP/CETSA, SPROX, and LiP‐SMAP, which monitor changes in the protein properties caused by ligand binding (Kosmacz et al., ; Ong et al., ; Piazza et al., ; Savitski et al., ; West, Tang, & Fitzgerald, ). These properties can be thermal stability of a protein, oxidation rate, or susceptibility of a protein to proteolysis.…”

Section: Commentarymentioning

confidence: 99%

PROMIS: Global Analysis of PROtein‐Metabolite Interactions

et al. 2019

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Small molecules are not only intermediates of metabolism, but also play important roles in signaling and in controlling cellular metabolism, growth, and development. Although a few systematic studies have been conducted, the true extent of protein–small molecule interactions in biological systems remains unknown. PROtein–metabolite interactions using size separation (PROMIS) is a method for studying protein–small molecule interactions in a non‐targeted, proteome‐ and metabolome‐wide manner. This approach uses size‐exclusion chromatography followed by proteomics and metabolomics liquid chromatography–mass spectrometry analysis of the collected fractions. Assuming that small molecules bound to proteins would co‐fractionate together, we found numerous small molecules co‐eluting with proteins, strongly suggesting the formation of stable complexes. Using PROMIS, we identified known small molecule–protein complexes, such as between enzymes and cofactors, and also found novel interactions. © 2019 The Authors. Basic Protocol 1: Preparation of native cell lysate from plant material Support Protocol: Bradford assay to determine protein concentration Basic Protocol 2: Separation of molecular complexes using size‐exclusion chromatography Basic Protocol 3: Simultaneous extraction of proteins and metabolites using single‐step extraction protocol Basic Protocol 4: Metabolomics analysis Basic Protocol 5: Proteomics analysis

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Interaction of 2',3'-cAMP with Rbp47b plays a role in stress granule formation

Cited by 39 publications

References 49 publications

Protein and metabolite composition of Arabidopsis stress granules

Protein and metabolite composition of Arabidopsis stress granules

Simultaneous Inhibition of Glycolysis and Oxidative Phosphorylation Triggers a Multi-Fold Increase in Secretion of Exosomes: Possible Role of 2′,3′-cAMP

PROMIS: Global Analysis of PROtein‐Metabolite Interactions

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