Phosphatidic acid (PA) and phosphoinositides are metabolically interconverted lipid second messengers that have central roles in many growth factor (GF)-stimulated signalling pathways. Yet, little is known about the mechanisms that coordinate their production and downstream signalling. Here we show that the phosphatidylinositol (PI)-transfer protein Nir2 translocates from the Golgi complex to the plasma membrane in response to GF stimulation. This translocation is triggered by PA formation and is mediated by its C-terminal region that binds PA in vitro. We further show that depletion of Nir2 substantially reduces the PI(4,5)P2 levels at the plasma membrane and concomitantly GF-stimulated PI(3,4,5)P3 production. Finally, we show that Nir2 positively regulates the MAPK and PI3K/AKT pathways. We propose that Nir2 through its PA-binding capability and PI-transfer activity can couple PA to phosphoinositide signalling, and possibly coordinates their local lipid metabolism and downstream signalling.

The functional rules for microRNA (miRNA) targeting remain controversial despite their biological importance because only a small fraction of distinct interactions, called site types, have been examined among an astronomical number of site types that can occur between miRNAs and their target mRNAs. To systematically discover functional site types and to evaluate the contradicting rules reported previously, we used large-scale transcriptome data and statistically examined whether each of approximately 2 billion site types is enriched in differentially downregulated mRNAs responding to overexpressed miRNAs. Accordingly, we identified seven non-canonical functional site types, most of which are novel, in addition to four canonical site types, while also removing numerous false positives reported by previous studies. Extensive experimental validation and significantly elevated 3' UTR sequence conservation indicate that these non-canonical site types may have biologically relevant roles. Our expanded catalog of functional site types suggests that the gene regulatory network controlled by miRNAs may be far more complex than currently understood.

The integral endoplasmic reticulum (ER)-membrane protein VAP-B interacts with various lipid-transfer/binding proteins containing an FFAT motif through its N-terminal MSP domain. A genetic mutation within its MSP domain, P56S, was identified in familial forms of motor neuron diseases. This mutation induces the formation of insoluble VAP-B(P56S) protein aggregates by an unknown mechanism. In this study, we defined the structural requirements for VAP-B oligomerization and demonstrated their contribution for VAP-B(P56S) aggregation and neurotoxicity. We show that the oligomerization of VAP-B is mainly mediated by its coiled-coil domain and that the GXXXG dimerization motif within the transmembrane domain mediates transmembrane domains self-association but is insufficient to drive VAP-B oligomerization. We further show that the oligomerization of the wild-type VAP-B is independent of its MSP domain. However, we found that the P56S mutation induces conformational changes within the MSP domain and facilitates its propensity to aggregate by exposing hydrophobic patches to the solvent. These conformational changes have no direct effect on FFAT binding. Rather, they enhance VAP-B(P56S) oligomerization driven by the combined contributions of the coiled-coil and the transmembrane domains, thereby preventing accessibility to FFAT-binding site, facilitating the production of VAP-B(P56S)-insoluble aggregates and consequently its neurotoxicity. These results shed light on the mechanism by which VAP-B(P56S) aggregates are formed and induce familial motor neuron diseases.Protein aggregation and inclusion body formation are common pathological features of many neurological disorders including Huntington disease, Alzheimer disease, Parkinson disease, and amyotrophic lateral sclerosis (ALS) 3 (1, 2).ALS is a fatal neurodegenerative disorder involving progressive loss of motor neurons in the cerebral cortex, brain stem, and spinal cord (3). Approximately 10% of the cases are familial, of which 20% are caused by dominantly inherited mutations in the Cu/Zn-superoxide dismutase (SOD) gene (4, 5). Recently, mutations in other genes, including the human VAP-B, have been identified in ALS patients (6 -8).The mutation in VAP-B substitutes a highly conserved proline residue at position 56 by a serine (P56S) and is associated with three forms of human motor neuron diseases: a late-onset spinal muscular atrophy, an atypical ALS type 8, and a typical severe ALS with rapid progression (7, 9). Atypical ALS type 8 is an autosomal dominant slowly progressive disorder characterized by fasciculation, cramps, and postural tremor. It is currently unknown how this P56S mutation induces motor neuron degeneration. Nevertheless, recent studies suggest that the VAP-B(P56S) mutant acts in a dominant fashion through dimerization with the wild-type protein and inactivating the heterodimer and that expression of the VAP-B(P56S) mutant induces the formation of insoluble protein aggregates (7, 10 -15). Indeed, co-expression studies suggest that wild-type VAP-B is...

The involvement of epithelial-mesenchymal transition (EMT) in breast cancer metastasis has been demonstrated in many studies. However, the intracellular proteins and signaling pathways that regulate EMT have not been fully identified. Here, we show that the lipid-transfer protein Nir2 (also known as PITPNM1) enhances EMT in mammary epithelial and breast cancer cells. Nir2 overexpression decreases the expression of epithelial markers and concomitantly increases the expression of mesenchymal markers, whereas silencing of Nir2 expression by small hairpin RNA (shRNA) has opposite effects. Additionally, Nir2 expression is increased during EMT and affects cell morphology, whereas Nir2 depletion attenuates growth factor-induced cell migration. These effects of Nir2 on EMTassociated processes are mainly mediated through the PI3K/AKT and the ERK1/2 pathways. Nir2 depletion also inhibits cell invasion in vitro and lung metastasis in animal models. Immunohistochemical analysis of breast cancer tissue samples reveals a correlation between high Nir2 expression and tumor grade, and Kaplan-Meier survival curves correlate Nir2 expression with poor disease outcome. These results suggest that Nir2 not only enhances EMT in vitro and breast cancer metastasis in animal models, but also contributes to breast cancer progression in human patients.

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