Yeast Two‐Hybrid Assay to Identify Interacting Proteins

Paiano, Aurora; Margiotta, Azzurra; Luca, Maria De; Bucci, Cecilia

doi:10.1002/cpps.70

Cited by 65 publications

(37 citation statements)

References 12 publications

(14 reference statements)

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“…These proteins activate the AF-2 domain of the nuclear receptor and enhance transcription by linking the liganded nuclear receptor to the basal transcription machinery. We have identified almost all these groups of coregulators using either a direct protein-protein interaction assay, such as a yeast two-hybrid assay [150], GST-pull downs [124], and ligand affinity chromatography [141] to identify the PPARα-interacting proteins and a functional transcriptional activation complex [131]. PPARs, like other nuclear receptors, interact with coactivators such as SRC-1 (steroid receptor coactivator-1) or corepressors such as NCoR and SMRT.…”

Section: Pparα and Coregulatorsmentioning

confidence: 99%

Mechanisms Mediating the Regulation of Peroxisomal Fatty Acid Beta-Oxidation by PPARα

Tahri-Joutey

Andreoletti

Surapureddi

et al. 2021

IJMS

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In mammalian cells, two cellular organelles, mitochondria and peroxisomes, share the ability to degrade fatty acid chains. Although each organelle harbors its own fatty acid β-oxidation pathway, a distinct mitochondrial system feeds the oxidative phosphorylation pathway for ATP synthesis. At the same time, the peroxisomal β-oxidation pathway participates in cellular thermogenesis. A scientific milestone in 1965 helped discover the hepatomegaly effect in rat liver by clofibrate, subsequently identified as a peroxisome proliferator in rodents and an activator of the peroxisomal fatty acid β-oxidation pathway. These peroxisome proliferators were later identified as activating ligands of Peroxisome Proliferator-Activated Receptor α (PPARα), cloned in 1990. The ligand-activated heterodimer PPARα/RXRα recognizes a DNA sequence, called PPRE (Peroxisome Proliferator Response Element), corresponding to two half-consensus hexanucleotide motifs, AGGTCA, separated by one nucleotide. Accordingly, the assembled complex containing PPRE/PPARα/RXRα/ligands/Coregulators controls the expression of the genes involved in liver peroxisomal fatty acid β-oxidation. This review mobilizes a considerable number of findings that discuss miscellaneous axes, covering the detailed expression pattern of PPARα in species and tissues, the lessons from several PPARα KO mouse models and the modulation of PPARα function by dietary micronutrients.

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Section: Pparα and Coregulatorsmentioning

confidence: 99%

Mechanisms Mediating the Regulation of Peroxisomal Fatty Acid Beta-Oxidation by PPARα

Tahri-Joutey

Andreoletti

Surapureddi

et al. 2021

IJMS

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“…The construct pGBKT7-HrpAM was used to screen a HeLa cell line cDNA library cloned in the pGADGH vector and a human fetal liver cDNA library cloned in the pACT-2 vector, using AH109 yeast strain as a host [ 17 , 18 , 20 , 21 ]. Transformants were plated onto SD medium lacking histidine, leucine and tryptophan.…”

Section: Methodsmentioning

confidence: 99%

HrpA anchors meningococci to the dynein motor and affects the balance between apoptosis and pyroptosis

et al. 2022

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Background In Neisseria meningitidis the HrpA/HrpB two-partner secretion system (TPS) was implicated in diverse functions including meningococcal competition, biofilm formation, adherence to epithelial cells, intracellular survival and vacuolar escape. These diverse functions could be attributed to distinct domains of secreted HrpA. Methods A yeast two-hybrid screening, in vitro pull-down assay and immunofluorescence microscopy experiments were used to investigate the interaction between HrpA and the dynein light-chain, Tctex-type 1 (DYNLT1). In silico modeling was used to analyze HrpA structure. Western blot analysis was used to investigate apoptotic and pyroptotic markers. Results The HrpA carboxy-terminal region acts as a manganese-dependent cell lysin, while the results of a yeast two-hybrid screening demonstrated that the HrpA middle region has the ability to bind the dynein light-chain, Tctex-type 1 (DYNLT1). This interaction was confirmed by in vitro pull-down assay and immunofluorescence microscopy experiments showing co-localization of N. meningitidis with DYNLT1 in infected epithelial cells. In silico modeling revealed that the HrpA-M interface interacting with the DYNLT1 has similarity with capsid proteins of neurotropic viruses that interact with the DYNLT1. Indeed, we found that HrpA plays a key role in infection of and meningococcal trafficking within neuronal cells, and is implicated in the modulation of the balance between apoptosis and pyroptosis. Conclusions Our findings revealed that N. meningitidis is able to effectively infect and survive in neuronal cells, and that this ability is dependent on HrpA, which establishes a direct protein–protein interaction with DYNLTI in these cells, suggesting that the HrpA interaction with dynein could be fundamental for N. meningitidis spreading inside the neurons. Moreover, we found that the balance between apoptotic and pyroptotic pathways is heavily affected by HrpA.

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“…Untransformed Saccharomyces cerevisiae L40 was propagated in yeast extract-peptone-dextrose (YEPD) medium supplemented with 0.2% adenine and grown at 30°C. Yeast two-hybrid bait and prey plasmids were cotransformed into strain L40 using the lithium acetate/polyethylene glycol (PEG) protocol ( 35 ), and cotransformants were selected using SD -Trp/-Leu dropout medium (synthetic defined medium lacking tryptophan or leucine). Liquid β-galactosidase Miller assays were performed as described previously ( 35 ), except that colonies were taken directly from SD -Trp/-Leu plates after growth for 3 or 4 days and resuspended in media for use in the assay.…”

Section: Methodsmentioning

confidence: 99%