We reveal the identification of a polybasic motif necessary for polyphosphoinositide interaction and nucleolar targeting of ErbB3 binding protein 1 (EBP1). EBP1 interacts directly with phosphatidylinositol(3,4,5)-triphosphate and their association is detected in the nucleolus, implying regulatory roles of nucleolar processes.

Lipid nanodiscs influence α-Syn oligomerization rates M. Jakubec et al.

Polyphosphoinositides (PPIns) play essential roles as lipid signaling molecules, and many of their functions have been elucidated in the cytoplasm. However, PPIns are also intranuclear where they contribute to chromatin remodeling, transcription, and mRNA splicing. The PPIn, phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5) P 3 ), has been mapped to the nucleus and nucleoli, but its role remains unclear in this subcellular compartment. To gain further insights into the nuclear functions of PtdIns(3,4,5) P 3 , we applied a previously developed quantitative MS-based approach to identify the targets of PtdIns(3,4,5) P 3 from isolated nuclei. We identified 179 potential PtdIns(3,4,5) P 3 -interacting partners, and gene ontology analysis for the biological functions of this dataset revealed an enrichment in RNA processing/splicing, cytokinesis, protein folding, and DNA repair. Interestingly, about half of these interactors were common to nucleolar protein datasets, some of which had dual functions in rRNA processes and DNA repair, including poly(ADP-ribose) polymerase 1 (PARP1, now referred as ADP-ribosyltransferase 1). PARP1 was found to interact directly with PPIn via three polybasic regions in the DNA-binding domain and the linker located N-terminal of the catalytic region. PARP1 was shown to bind to PtdIns(3,4,5) P 3 as well as phosphatidylinositol 3,4-bisphosphate in vitro and to colocalize with PtdIns(3,4,5) P 3 in the nucleolus and with phosphatidylinositol 3,4-bisphosphate in nucleoplasmic foci. In conclusion, the PtdIns(3,4,5) P 3 interactome reported here will serve as a resource to further investigate the molecular mechanisms underlying PtdIns(3,4,5) P 3 -mediated interactions in the nucleus and nucleolus.

Dysregulation of the biosynthesis of cholesterol and other lipids has been implicated in neurological diseases, including Parkinson's disease, where the misfolding of membraneassociated α-Synuclein is a key molecular event. Recent research also suggests that α-Synuclein aggregation is influenced by the lipid environment. The exact molecular mechanisms responsible for cholesterol’s effect on α-Synuclein binding to lipids and how this binding may affect α-Synuclein oligomerization and fibrillation remain elusive, as does the relative importance of cholesterol versus other lipid factors. We probed the interactions and fibrillation behaviour of α-Synuclein using SMA nanodiscs, containing zwitterionic and anionic lipid model systems with and without cholesterol. SPR and ThT fluorescence assays were then employed to monitor α-Synuclein binding, as well as fibrillation in the absence and presence of membrane models. 1H-15N correlated NMR was used to monitor the fold of α-Synuclein in response to nanodisc binding, and we determined individual residue apparent affinities for the nanodisc-contained bilayers. Cholesterol inhibited α-Synuclein interaction with lipid bilayers. We also find that cholesterol significantly promotes α-Synuclein fibrillation, with a more than 20-fold reduction of lag-times before fibrillation onset. When α-Synuclein-bilayer interactions were analysed for individual residues by solution-state NMR, we observed two different effects of cholesterol. In nanodiscs made of DOPC, cholesterol modulated the NAC part of α-Synuclein, leading to stronger interaction of this region with the lipid bilayer. In contrast, in the nanodiscs comprising DOPC, DOPE and DOPG, the NAC part was mostly unaffected by cholesterol, while the binding of the N-terminal and the C-terminal were both inhibited.

The class I phosphoinositide 3-kinase (PI3K) catalytic subunits p110α and p110β are ubiquitously expressed but differently targeted in tumours. In cancer, PIK3CB (encoding p110β) is seldom mutated compared to PIK3CA (encoding p110α) but can contribute to tumorigenesis in certain PTEN-deficient tumours. The underlying molecular mechanisms are however unclear. We have previously reported that p110β is highly expressed in endometrial cancer (EC) cell lines and at the mRNA levels in primary patient tumours. Here, we show that p110β protein levels are high in both the cytoplasmic and nuclear compartments in EC cells. Moreover, high nuclear/cytoplasmic staining ratio were detected in high grade primary tumours. High levels of phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) were measured in the nucleus of EC cells and pharmacological and genetic approaches showed that its production was partly dependent upon p110β activity. Using immunofluorescence staining, p110β and PtdIns(3,4,5)P3 were localised in the nucleolus, which correlated with high levels of 47S pre-rRNA. p110β inhibition led to a decrease in 47S rRNA levels and cell proliferation. In conclusion, these results present a nucleolar role for p110β that may contribute to tumorigenesis in EC.