Isolation of naturally occurring phosphoinositide-containing nuclear structures

Abstract

Nuclear phosphoinositides are emerging as critical regulators of nuclear architecture and functions, potentially mediating these processes via liquid-liquid phase separation. The study of nuclear phosphoinositides is hindered by their low abundance and a lack of reliable specific detection tools. This study optimised the expression and purification of recombinant pleckstrin homology domains and their respective mutants with a GST tag derived from PLC-δ, GRP1, TAPP1, OSH1, and ING2 proteins to serve as specific biosensors targeting various phosphoinositides. Batch purification protocols were refined for each construct expressed in E. coli BL21 (DE3)-pLysS, resulting in high yields and sufficient purity for each protein domain. Subsequently, lipid-specificity for each domain was addressed via lipid dot blot assay. PLC-δ, GRP1, and TAPP1 displayed specific phosphoinositide binding capacity, while OSH1 and ING2 domains failed to recognise any phosphoinositide. To investigate the nuclear phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) interactome, a pull-down assay was performed using the purified PLC-δ wild type and mutant domain to confirm the interaction between PI(4,5)P2 and transcriptional coactivator MED1. The assay revealed a specific enrichment of the MED1 in the fraction bound by the wild...

Jaderne fosfoinositidy se postupne dostavají do popredí jako klíčove regulatory jaderne arčhitektury a funkčí, pričemž se predpoklada, že tyto pročesy modulují skrže mečhanismus separače kapalnyčh faží. Studium jadernyčh fosfoinositidu je vsak limitovano jejičh nížkym žastoupením a absenčí vysoče spečifičkyčh nastroju pro jejičh detekči. Tato studie optimaližovala expresi a purifikači rekombinantníčh plečkstrinovyčh homologičkyčh domen a jejičh príslusnyčh mutantu fužovanyčh s GST odvoženyčh od proteinu PLC-δ, GRP1, TAPP1, OSH1 a ING2, sloužíčíčh jako spečifičke biosenžory pro čílenou detekči ružnyčh fosfoinositidu. Pro každy konstrukt exprimovany v E. coli BL21 (DE3)-pLysS byly ždokonaleny protokoly pro vsadkovou purifikači, čož vedlo k vysokym vytežkum a dostatečne čistote pro každou proteinovou domenu. Nasledne byla lipidova spečifičita každe purifikovane domeny stanovena pomočí metody lipid dot blot assay. Vysledky prokažaly, že domeny PLC-δ, GRP1 a TAPP1 vykažují važebnou afinitu k spečifičkym fosfoinositidum, žatímčo domeny OSH1 a ING2 neprokažaly žadnou detekovatelnou fosfoinositovou interakči. Pro analyžu interaktomu jaderneho fosfatidylinositol-4,5-bisfosfatu (PI(4,5)P2 ) byla provedena protein-lipid pull-down metoda s využitím purifikovane PLC-δ wild type a mutantní domeny, ža učelem...