Attenuation of PITPNM1 Signaling Cascade Can Inhibit Breast Cancer Progression

Phosphatidylinositol transfer protein membrane-associated 1 (PITPNM1) contains a highly conserved phosphatidylinositol transfer domain which is involved in phosphoinositide trafficking and signaling transduction under physiological conditions. However, the functional role of PITPNM1 in cancer progression remains unknown. Here, by integrating datasets of The Cancer Genome Atlas (TCGA) and Molecular Taxonomy of Breast Cancer (METABRIC), we found that the expression of PITPNM1 is much higher in breast cancer tissues than in normal breast tissues, and a high expression of PITPNM1 predicts a poor prognosis for breast cancer patients. Through gene set variation analysis (GSEA) and gene ontology (GO) analysis, we found PITPNM1 is mainly associated with carcinogenesis and cell-to-cell signaling ontology. Silencing of PITPNM1, in vitro, significantly abrogates proliferation and colony formation of breast cancer cells. Collectively, PITPNM1 is an important prognostic indicator and a potential therapeutic target for breast cancer.

Early Changes in Skeletal Muscle of Young C22 Mice, A Model of Charcot-Marie-Tooth 1A

Background: The C22 mouse is a Charcot-Marie-Tooth 1A transgenic model with minimal axonal loss. Objective: To analyse early skeletal muscle changes resulting from this dysmyelinating neuropathy. Methods: Histology of tibialis anterior muscles of C22 mice and wild type litter mate controls for morphometric analysis and (immuno-)histochemistry for known denervation markers and candidate proteins identified by representational difference analysis (RDA) based on mRNA from the same muscles; quantitative PCR and Western blotting for confirmation of RDA findings. Results: At age 10 days, morphometry was not different between groups, while at 21 days, C22 showed significantly more small diameter fibres, indicating the onset of atrophy at an age when weakness becomes detectable. Neither (immuno-)histochemistry nor RDA detected extrajunctional expression of acetylcholine receptors by age 10 and 21 days, respectively. RDA identified some mRNA up-regulated in C22 muscles, among them at 10 days, prior to detectable weakness or atrophy, integral membrane protein 2a (Itm2a), eukaryotic initiation factor 2, subunit 2 (Eif2s2) and cytoplasmic phosphatidylinositol transfer protein 1 (Pitpnc1). However, qPCR failed to measure significant differences. In contrast, Itm2a and Eif2s2 mRNA were significantly down-regulated comparing 21 versus 10 days of age in both groups, C22 and controls. Western blotting confirmed significant down-regulation of ITM2A protein in C22 only. Conclusion: Denervation-like changes in this model develop slowly with onset of atrophy and weakness at about three weeks of age, before detection of extrajunctional acetylcholine receptors. Altered Itm2a expression seems to being early as an increase, but becomes distinct as a decrease later.

CRISPR/Cas9 mediated somatic gene therapy for insertional mutations: the vibrator mouse model

Somatic gene therapy remains technically challenging, especially in the central nervous system (CNS). Efficiency of gene delivery, efficacy in recipient cells, and proportion of cells required for overall benefit are the key points needed to be considered in any therapeutic approach. Recent efforts have demonstrated the efficacy of RNA-guided nucleases such as CRISPR/Cas9 in correcting point mutations or removing dominant mutations. Here we used viral delivered Cas9 plasmid and two guide RNAs to remove a recessive insertional mutation, vibrator (vb), in the mouse brain. vb mice express ∼20% of normal levels of phosphatidylinositol transfer α (Pitpna) RNA and protein due to an endogenous retrovirus inserted in intron 4, resulting in early-onset tremor, degeneration of brainstem and spinal cord neurons, and juvenile death. The in situ CRISPR/Cas9 viral treatment effectively delayed neurodegeneration, attenuated tremor, and bypassed juvenile death. Our studies demonstrate the potential of CRISPR/Cas9-mediated gene therapy for insertional mutations in the postnatal brain.

βA3/A1-crystallin regulates apical polarity and EGFR endocytosis in retinal pigmented epithelial cells

The retinal pigmented epithelium (RPE) is a monolayer of multifunctional cells located at the back of the eye. High membrane turnover and polarization, including formation of actin-based apical microvilli, are essential for RPE function and retinal health. Herein, we demonstrate an important role for βA3/A1-crystallin in RPE. βA3/A1-crystallin deficiency leads to clathrin-mediated epidermal growth factor receptor (EGFR) endocytosis abnormalities and actin network disruption at the apical side that result in RPE polarity disruption and degeneration. We found that βA3/A1-crystallin binds to phosphatidylinositol transfer protein (PITPβ) and that βA3/A1-crystallin deficiency diminishes phosphatidylinositol 4,5-biphosphate (PI(4,5)P2), thus probably decreasing ezrin phosphorylation, EGFR activation, internalization, and degradation. We propose that βA3/A1-crystallin acquired its RPE function before evolving as a structural element in the lens, and that in the RPE, it modulates the PI(4,5)P2 pool through PITPβ/PLC signaling axis, coordinates EGFR activation, regulates ezrin phosphorylation and ultimately the cell polarity.

Emerging Prospects for Combating Fungal Infections by Targeting Phosphatidylinositol Transfer Proteins

The emergence of fungal “superbugs” resistant to the limited cohort of anti-fungal agents available to clinicians is eroding our ability to effectively treat infections by these virulent pathogens. As the threat of fungal infection is escalating worldwide, this dwindling response capacity is fueling concerns of impending global health emergencies. These developments underscore the urgent need for new classes of anti-fungal drugs and, therefore, the identification of new targets. Phosphoinositide signaling does not immediately appear to offer attractive targets due to its evolutionary conservation across the Eukaryota. However, recent evidence argues otherwise. Herein, we discuss the evidence identifying Sec14-like phosphatidylinositol transfer proteins (PITPs) as unexplored portals through which phosphoinositide signaling in virulent fungi can be chemically disrupted with exquisite selectivity. Recent identification of lead compounds that target fungal Sec14 proteins, derived from several distinct chemical scaffolds, reveals exciting inroads into the rational design of next generation Sec14 inhibitors. Development of appropriately refined next generation Sec14-directed inhibitors promises to expand the chemical weaponry available for deployment in the shifting field of engagement between fungal pathogens and their human hosts.

Interdomain interactions regulate the localization of a lipid transfer protein at ER-PM contact sites

ABSTRACT During phospholipase C-β (PLC-β) signalling in Drosophila photoreceptors, the phosphatidylinositol transfer protein (PITP) RDGB, is required for lipid transfer at endoplasmic reticulum (ER)–plasma membrane (PM) contact sites (MCS). Depletion of RDGB or its mis-localization away from the ER–PM MCS results in multiple defects in photoreceptor function. Previously, the interaction between the FFAT motif of RDGB and the integral ER protein dVAP-A was shown to be essential for accurate localization to ER–PM MCS. Here, we report that the FFAT/dVAP-A interaction alone is insufficient to localize RDGB accurately; this also requires the function of the C-terminal domains, DDHD and LNS2. Mutations in each of these domains results in mis-localization of RDGB leading to loss of function. While the LNS2 domain is necessary, it is not sufficient for the correct localization of RDGB, which also requires the C-terminal DDHD domain. The function of the DDHD domain is mediated through an intramolecular interaction with the LNS2 domain. Thus, interactions between the additional domains in a multi-domain PITP together lead to accurate localization at the MCS and signalling function. This article has an associated First Person interview with the first author of the paper.

Translational control of lipogenesis links protein synthesis and phosphoinositide signaling with nuclear division

ABSTRACTContinuously dividing cells coordinate their growth and division. How fast cells grow in mass determines how fast they will multiply. Yet, there are few, if any, examples of a metabolic pathway that actively drives a cell cycle event instead of just being required for it. Here, we show that translational upregulation of lipogenic enzymes in yeast increased the abundance of lipids and accelerated nuclear elongation and division. De-repressing translation of acetyl CoA carboxylase and fatty acid synthase also suppressed cell cycle-related phenotypes, including delayed nuclear division, associated with Sec14p phosphatidylinositol transfer protein deficiencies, and the irregular nuclear morphologies of mutants defective in phosphatidylinositol 4-OH kinase activities. Our results show that increased lipogenesis drives a critical cell cycle landmark and report a phosphoinositide signaling axis in control of nuclear division. The broad conservation of these lipid metabolic and signaling pathways raises the possibility these activities similarly govern nuclear division in mammals.