Biology subjects

Lewis, A. E.

Publications and source records attributed to Lewis, A. E..

4 recordsLinked to original sources

bioRxiv2025

Lysine-rich regions located in the C-terminal domain of DNA Topoisomerase 2-alpha act as polyphosphoinositide interaction sites and nucleolar localisation signals

Polyphosphoinositides (PPIn) are lipid signalling molecules that regulate essential cellular processes in eukaryotic cells by regulating effector proteins not only in the cytoplasm, but also in the nucleus. To decipher PPIn nuclear functions, we have previously established quantitative proteomic methods in combination with PPIn pull-down to identify nuclear PPIn-binding proteins. We focused our analyses on one of these proteins, DNA Topoisomerase 2 (TOP2A), an enzyme known to resolve DNA topological problems occurring during replication, transcription and mitosis. In this study, we validated its direct interaction with PPIn and identified the binding site consisting of two lysine-rich polybasic regions (PBR) located within its C-terminal domain (CTD) at residues 1228-1237 and 1259-1276. Overexpression studies of the WT and PBR deletion mutants showed that these two PBR were required for TOP2A nucleolar localisation. TOP2A is localised to different sub-nuclear sites where PPIn have also been detected. We hence investigated the localisation of TOP2A by immunofluorescence microscopy and cell fractionation in relation to PPIn. We show that TOP2A colocalised with the PPIn phosphatidylinositol(4,5)bisphosphate (PtdIns(4,5)P2) in nuclear speckles of transcriptionally arrested HeLa cells. Inhibition of type II phosphatidylinositol-5-phosphate 4-kinase, which generates PtdIns(4,5)P2 from PtdIns5P, led to an increase of TOP2A levels associated with the chromatin. Our results demonstrated TOP2A as a PPIn effector protein and a role for nuclear PPIn in regulating the subnuclear dynamic of TOP2A.

molecular biology↗

bioRxiv2023

Exposure to early life stress impairs weight loss maintenance success in mice.

Early life stress increases obesity risk, but its impact on weight loss maintenance is unknown. Mice underwent neonatal maternal separation (NMS) from 0-3 weeks and were weaned onto high fat sucrose diet (HFSD) from 3-20 weeks. Calorie-restricted weight loss on a low fat sucrose diet (LFSD) occurred over 2 weeks to induce a 20% loss in body weight, which was maintained for 6 weeks. After weight loss, half the mice received running wheels (EX) the other half remained sedentary (SED). Mice were then fed ad libitum on HFSD or LFSD for 10 weeks and allowed to regain body weight. NMS mice had greater weight regain, total body weight and adiposity compared to naive mice. During the first week of refeeding, NMS mice had increased food intake and were in a greater positive energy balance than naive mice, but total energy expenditure was not affected by NMS. Female mice were more susceptible to NMS-induced effects, including increases in adiposity. NMS and naive females were more susceptible to HFSD-induce weight regain. Exercise was beneficial in the first week of regain in male mice, but long-term only those on LFSD benefited from EX. As expected, HFSD led to greater weight regain than LFSD.

physiology↗

bioRxiv2020

Nuclear phosphatidylinositol 3,4,5-trisphosphate interactome uncovers an enrichment in nucleolar proteins

Polyphosphoinositides (PPIn) play essential functions as lipid signalling molecules and many of their functions have been elucidated in the cytoplasm. However, PPIn are also intranuclear where they contribute to chromatin remodelling, transcription and mRNA splicing. The PPIn, phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) 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)P3, we applied a previously developed quantitative mass spectrometry-based approach to identify the targets of PtdIns(3,4,5)P3 from isolated nuclei. We identified 179 potential PtdIns(3,4,5)P3-interacting proteins 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 transcription and DNA repair, including Poly(ADP-Ribose) Polymerase 1 (PARP1/ARTD1). PARP1 was found to interact directly with PtdIns(3,4,5)P3 as well as PtdIns(3,4)P2 and to co-localise with PtdIns(3,4,5)P3 in the nucleolus and with PtdIns(3,4)P2 in nucleoplasmic foci. In conclusion, the PtdIns(3,4,5)P3 interactome reported here identified several nucleolar proteins and further pointed to roles for this lipid in these processes.

molecular biology↗

bioRxiv2019

Nuclear upregulation of PI3K p110β correlates with increased rRNA transcription in endometrial cancer cells

Genes encoding for components of the phosphoinositide 3-kinase (PI3K) pathway are frequently mutated in cancer, including inactivating mutations of PTEN and activating mutations of PIK3CA, encoding the PI3K catalytic subunit p110. PIK3CB, encoding p110{beta}, is rarely mutated, but can contribute to tumourigenesis in some PTEN-deficient tumours. The underlying molecular mechanisms are however poorly understood. By analysing cell lines and annotated clinical samples, we have previously found that p110{beta} is highly expressed in endometrial cancer (EC) cell lines and that PIK3CB mRNA levels increase early in primary tumours correlating with lower survival. Selective inhibition of p110 and p110{beta} led to different effects on cell signalling and cell function, p110 activity being correlated to cell survival in PIK3CA mutant cells and p110{beta} with cell proliferation in PTEN-deficient cells. To understand the mechanisms governing the differential roles of these isoforms, we assessed their sub-cellular localisation. p110 was cytoplasmic whereas p110{beta} was both cytoplasmic and nuclear with increased levels in both compartments in cancer cells. Immunohistochemistry of p110{beta} in clinically annotated patient tumour sections revealed high nuclear/cytoplasmic staining ratio, which correlated significantly with higher grades. Consistently, the presence of high levels of p110{beta} in the nuclei of EC cells, correlated with high levels of its product phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) in the nucleus. Using immunofluorescence labelling, we observed both p110{beta} and PtdIns(3,4,5)P3 in the nucleoli of EC cell lines. The production of nucleolar PtdIns(3,4,5)P3 was dependent upon p110{beta} activity. EC cells with high levels of nuclear PtdIns(3,4,5)P3 and p110{beta} showed elevated nucleolar activity as assessed by the increase in 47S pre-rRNA transcriptional levels in a p110{beta}-dependent manner. Altogether, these results present a nucleolar role for the PI3K pathway that may contribute to tumour progression in endometrial cancer.

molecular biology↗