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.

Phylogenetic analysis of plant multi-domain SEC14-like phosphatidylinositol transfer proteins and structure–function properties of PATELLIN2

Key message SEC14L-PITPs guide membrane recognition and signaling. An increasingly complex modular structure of SEC14L-PITPs evolved in land plants compared to green algae. SEC14/CRAL-TRIO and GOLD domains govern membrane binding specificity. Abstract SEC14-like phosphatidylinositol transfer proteins (SEC14L-PITPs) provide cues for membrane identity by exchanging lipophilic substrates, ultimately governing membrane signaling. Flowering plant SEC14L-PITPs often have modular structure and are associated with cell division, development, and stress responses. Yet, structure–function relationships for biochemical–cellular interactions of SEC14L-PITPs are rather enigmatic. Here, we evaluate the phylogenetic relationships of the SEC14L-PITP superfamily in the green lineage. Compared to green algae, land plants have an extended set of SEC14L-PITPs with increasingly complex modular structure. SEC14-GOLD PITPs, present in land plants but not Chara, diverged to three functional subgroups, represented by the six PATELLIN (PATL) proteins in Arabidopsis. Based on the example of Arabidopsis PATL2, we dissect the functional domains for in vitro binding to phosphoinositides and liposomes and for plant cell membrane association. While the SEC14 domain and its CRAL-TRIO-N-terminal extension serve general membrane attachment of the protein, the C-terminal GOLD domain directs it to the plasma membrane by recognizing specific phosphoinositides. We discuss that the different domains of SEC14L-PITPs integrate developmental and environmental signals to control SEC14L-PITP-mediated membrane identity, important to initiate dynamic membrane events.

Phosphatidylinositol transfer proteins regulate megakaryocyte TGF-β1 secretion and hematopoiesis in mice

Key Points PITPs facilitate TGF-β1 secretion in megakaryocytes regulating hematopoiesis. Pitpα −/− /β −/− megakaryocytes have a defect in α-granule morphology and function.

The Sec14-like phosphatidylinositol transfer proteins Sec14l3/SEC14L2 act as GTPase proteins to mediate Wnt/Ca2+ signaling

The non-canonical Wnt/Ca2+ signaling pathway plays important roles in embryonic development, tissue formation and diseases. However, it is unclear how the Wnt ligand-stimulated, G protein-coupled receptor Frizzled activates phospholipases for calcium release. Here, we report that the zebrafish/human phosphatidylinositol transfer protein Sec14l3/SEC14L2 act as GTPase proteins to transduce Wnt signals from Frizzled to phospholipase C (PLC). Depletion of sec14l3 attenuates Wnt/Ca2+ responsive activity and causes convergent and extension (CE) defects in zebrafish embryos. Biochemical analyses in mammalian cells indicate that Sec14l3-GDP forms complex with Frizzled and Dishevelled; Wnt ligand binding of Frizzled induces translocation of Sec14l3 to the plasma membrane; and then Sec14l3-GTP binds to and activates phospholipase Cδ4a (Plcδ4a); subsequently, Plcδ4a initiates phosphatidylinositol-4,5-bisphosphate (PIP2) signaling, ultimately stimulating calcium release. Furthermore, Plcδ4a can act as a GTPase-activating protein to accelerate the hydrolysis of Sec14l3-bound GTP to GDP. Our data provide a new insight into GTPase protein-coupled Wnt/Ca2+ signaling transduction.

Ligand and membrane-binding behavior of the phosphatidylinositol transfer proteins PITPα and PITPβ

Phosphatidylinositol transfer proteins (PITPs) are believed to be lipid transfer proteins because of their ability to transfer either phosphatidylinositol (PI) or phosphatidylcholine (PC) between membrane compartments, in vitro. However, the detailed mechanism of this transfer process is not fully established. To further understand the transfer mechanism of PITPs we examined the interaction of PITPs with membranes using dual polarization interferometry (DPI), which measures protein binding affinity on a flat immobilized lipid surface. In addition, a fluorescence resonance energy transfer (FRET)-based assay was also employed to monitor how quickly PITPs transfer their ligands to lipid vesicles. DPI analysis revealed that PITPβ had a higher affinity to membranes compared with PITPα. Furthermore, the FRET-based transfer assay revealed that PITPβ has a higher ligand transfer rate compared with PITPα. However, both PITPα and PITPβ demonstrated a preference for highly curved membrane surfaces during ligand transfer. In other words, ligand transfer rate was higher when the accepting vesicles were highly curved.