Regulatory Role of Phospholipids in Hepatitis C Virus Replication and Protein Function

Positive-strand RNA viruses such as hepatitis C virus (HCV) hijack key factors of lipid metabolism of infected cells and extensively modify intracellular membranes to support the viral lifecycle. While lipid metabolism plays key roles in viral particle assembly and maturation, viral RNA synthesis is closely linked to the remodeling of intracellular membranes. The formation of viral replication factories requires a number of interactions between virus proteins and host factors including lipids. The structure–function relationship of those proteins is influenced by their lipid environments and lipids that selectively modulate protein function. Here, we review our current understanding on the roles of phospholipids in HCV replication and of lipid–protein interactions in the structure–function relationship of the NS5A protein. NS5A is a key factor in membrane remodeling in HCV-infected cells and is known to recruit phosphatidylinositol 4-kinase III alpha to generate phosphatidylinositol 4-phosphate at the sites of replication. The dynamic interplay between lipids and viral proteins within intracellular membranes is likely key towards understanding basic mechanisms in the pathobiology of virus diseases, the mode of action of specific antiviral agents and related drug resistance mechanisms.

Adipocyte-specific deletion of PIP5K1c reduces diet-induced obesity and insulin resistance by increasing energy expenditure

Background Phosphatidylinositol 4-phosphate 5-kinase type I c (PIP5K1c) catalyses the synthesis of phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) by phosphorylating phosphatidylinositol 4 phosphate, which plays multiple roles in regulating focal adhesion formation, invasion, and cell migration signal transduction cascades. Here, a new physiological mechanism of PIP5K1c in adipocytes and systemic metabolism is reported. Methods Adipose-specific conditional knockout mice were generated to delete the PIP5K1c gene in adipocytes. In addition, in vitro research investigated the effect of PIP5K1c deletion on adipogenesis. Results Deletion of PIP5K1c in adipocytes significantly alleviated high fat diet (HFD)-induced obesity, hyperlipidaemia, hepatic steatosis, and insulin resistance. PIP5K1c deficiency in adipocytes also decreased adipocyte volume in HFD-induced obese mice, whereas no significant differences were observed in body weight and adipose tissue weight under normal chow diet conditions. PIP5K1c knockout in adipocytes significantly enhanced energy expenditure, which protected mice from HFD-induced weight gain. In addition, adipogenesis was markedly impaired in mouse stromal vascular fraction (SVF) from PIP5K1c-deleted mice. Conclusion Under HFD conditions, PIP5K1c regulates adipogenesis and adipose tissue homeostasis. Together, these data indicate that PIP5K1c could be a novel potential target for regulating fat accumulation, which could provide novel insight into the treatment of obesity.

Plasma membrane phosphatidylinositol-4-phosphate is not necessary for Candida albicans viability, yet is key for cell wall integrity and systemic infection

Phosphatidylinositol phosphates are key phospholipids with a range of regulatory roles, including membrane trafficking and cell polarity. Phosphatidylinositol-4-phosphate [PI(4)P] at the Golgi is required for the budding to filamentous growth transition in the human pathogenic fungus Candida albicans, however the role of plasma membrane PI(4)P is unclear. We have investigated the importance of this phospholipid in C. albicans growth, stress response, and virulence by generating mutant strains with decreased levels of plasma membrane PI(4)P, via deletion of components of the PI-4-kinase complex, i.e. Efr3, Ypp1 and Stt4. The amount of plasma membrane PI(4)P in the efr3∆/∆ and ypp1∆/∆ mutant was ~60% and ~40% of the wild-type strain, respectively, whereas it was nearly undetectable in the stt4∆/∆ mutant. All three mutants had reduced plasma membrane phosphatidylserine (PS). Although these mutants had normal yeast phase growth, they were defective in filamentous growth, exhibited defects in cell wall integrity and had an increased exposure of cell wall β(1,3)-glucan, yet they induced a range of hyphal specific genes. In a mouse model of hematogenously disseminated candidiasis, fungal plasma membrane PI(4)P levels directly correlated with virulence; the efr3∆/∆ had wild-type virulence, the ypp1∆/∆ mutant had attenuated virulence and the stt4∆/∆ mutant caused no lethality. In the mouse model of orpharyngeal candidiasis, only the ypp1∆/∆ mutant had reduced virulence, indicating that plasma membrane PI(4)P is less important for proliferation in the oropharynx. Collectively, these results demonstrate that plasma membrane PI(4)P levels play a central role in filamentation, cell wall integrity and virulence in C. albicans.

Genome-wide identification of PIP5K in wheat and its relationship with anther male sterility induced by high temperature

Background Phosphatidylinositol 4 phosphate 5-kinase (PIP5K) plays a key enzyme role in the inositol signal transduction system and has essential functions in plants in terms of growth, development, and stress responses. However, systematic studies on the wheat PIP5K gene family and its relation to male sterility have not been reported yet. Results Sixty-four TaPIP5K genes were identified. The TaPIP5K genes contained similar gene structures and conserved motifs on the same branches of the evolutionary tree, and their cis-regulatory elements were related to MeJA-responsiveness. Furthermore, 49 pairs of collinearity genes were identified and mainly subjected to purification selection during evolution. Synteny analyses showed that some PIP5K genes in wheat and the other four species shared a relatively conserved evolutionary process. The expression levels of many conservative TaPIP5K genes in HT-ms anthers were significantly lower than that in Normal anthers. In addition, HT-ms anthers have no dehiscence, and levels of OPDA and JA-ILE are significantly lower at the trinucleus stage. Conclusion These results indicate that the PIP5K gene family may be associated with male sterility induced by HT, and the reduction of JA-ILE levels and the abnormal levels of these genes expression may be one reason for the HT-ms anthers having no dehiscence, ultimately leading to the abortion of the anthers.

Imaging of Intracellular and Plasma Membrane Pools of PI(4,5)P2 and PI4P in Human Platelets

Phosphoinositides (PIs) are phosphorylated membrane lipids that have a plethora of roles in the cell, including vesicle trafficking, signaling, and actin reorganization. The most abundant PIs in the cell are phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] and phosphatidylinositol-4-monophosphate (PI4P). The localization and roles of both PI(4,5)P2 and PI4P are well established, is the broadly accepted methodological approach for their immunocytochemical visualization in different cell compartments in several cell lines. However, not much is known about these PIs in platelets (PLTs), the smallest blood cells that detect vessel wall injury, activate, and stop the bleeding. Therefore, we sought to investigate the localization of PI(4,5)P2 and PI4P in resting and activated PLTs by antibody staining. Here, we show that the intracellular pools of PI(4,5)P2 and PI4P can be detected by the established staining protocol, and these pools can be modulated by inhibitors of OCRL phosphatase and PI4KIIIα kinase. However, although resting PLTs readily stain for the plasma membrane (PM) pools of PI(4,5)P2 and PI4P, just a few activated cells were stained with the established protocol. We show that optimized protocol allows for the visualization of PI(4,5)P2 and PI4P at PM in activated PLTs, which could also be modulated by OCRL and PI4KIIIα inhibitors. We conclude that PI(4,5)P2 and PI4P are more sensitive to lipid extraction by permeabilizing agents in activated than in resting human PLTs, which suggests their different roles during PLT activation.

Membrane pools of phosphatidylinositol-4-phosphate regulate KCNQ1/KCNE1 membrane expression

Plasma membrane phosphatidylinositol 4-phosphate (PI4P) is a precursor of PI(4,5)P2, an important regulator of a large number of ion channels. Although the role of the phospholipid PI(4,5)P2 in stabilizing ion channel function is well established, little is known about the role of phospholipids in channel membrane localization and specifically the role of PI4P in channel function and localization. The phosphatidylinositol 4-kinases (PI4Ks) synthesize PI4P. Our data show that inhibition of PI4K and prolonged decrease of levels of plasma membrane PI4P lead to a decrease in the KCNQ1/KCNE1 channel membrane localization and function. In addition, we show that mutations linked to Long QT syndrome that affect channel interactions with phospholipids lead to a decrease in membrane expression. We show that expression of a LQT1-associated C-terminal deletion mutant abolishes PI4Kinase-mediated decrease in membrane expression and rescues membrane expression for phospholipid-targeting mutations. Our results indicate a novel role for PI4P on ion channel regulation. Our data suggest that decreased membrane PI4P availability to the channel, either due to inhibition of PI4K or as consequence of mutations, dramatically inhibits KCNQ1/KCNE1 channel membrane localization and current. Our results may have implications to regulation of other PI4P binding channels.

PI4P/PS countertransport by ORP10 at ER–endosome membrane contact sites regulates endosome fission

Membrane contact sites (MCSs) serve as a zone for nonvesicular lipid transport by oxysterol-binding protein (OSBP)-related proteins (ORPs). ORPs mediate lipid countertransport, in which two distinct lipids are transported counterdirectionally. How such lipid countertransport controls specific biological functions, however, remains elusive. We report that lipid countertransport by ORP10 at ER–endosome MCSs regulates retrograde membrane trafficking. ORP10, together with ORP9 and VAP, formed ER–endosome MCSs in a phosphatidylinositol 4-phosphate (PI4P)-dependent manner. ORP10 exhibited a lipid exchange activity toward its ligands, PI4P and phosphatidylserine (PS), between liposomes in vitro, and between the ER and endosomes in situ. Cell biological analysis demonstrated that ORP10 supplies a pool of PS from the ER, in exchange for PI4P, to endosomes where the PS-binding protein EHD1 is recruited to facilitate endosome fission. Our study highlights a novel lipid exchange at ER–endosome MCSs as a nonenzymatic PI4P-to-PS conversion mechanism that organizes membrane remodeling during retrograde membrane trafficking.

The Rpd3-Complex Regulates Expression of Multiple Cell Surface Recycling Factors in Yeast

Intracellular trafficking pathways control residency and bioactivity of integral membrane proteins at the cell surface. Upon internalisation, surface cargo proteins can be delivered back to the plasma membrane via endosomal recycling pathways. Recycling is thought to be controlled at the metabolic and transcriptional level, but such mechanisms are not fully understood. In yeast, recycling of surface proteins can be triggered by cargo deubiquitination and a series of molecular factors have been implicated in this trafficking. In this study, we follow up on the observation that many subunits of the Rpd3 lysine deacetylase complex are required for recycling. We validate ten Rpd3-complex subunits in recycling using two distinct assays and developed tools to quantify both. Fluorescently labelled Rpd3 localises to the nucleus and complements recycling defects, which we hypothesised were mediated by modulated expression of Rpd3 target gene(s). Bioinformatics implicated 32 candidates that function downstream of Rpd3, which were over-expressed and assessed for capacity to suppress recycling defects of rpd3∆ cells. This effort yielded three hits: Sit4, Dit1 and Ldb7, which were validated with a lipid dye recycling assay. Additionally, the essential phosphatidylinositol-4-kinase Pik1 was shown to have a role in recycling. We propose recycling is governed by Rpd3 at the transcriptional level via multiple downstream target genes.

Differential expression of PI4KA in cancer of the vulva.

In these brief notes we document work using published microarray data (1, 2) to pioneer integrative transcriptome analysis comparing vulvar carcinoma to its tissue of origin, the vulva. We report the differential expression of phosphatidylinositol 4-kinase alpha, encoded by PI4KA, in cancer of the vulva. PI4KA may be of pertinence to understanding transformation and disease progression in vulvar cancer (3).