Phosphatidylinositol Transfer Protein Research Articles

Genome-Wide Association Study on Imputed Genotypes of 180 Eurasian Soybean Glycine max Varieties for Oil and Protein Contents in Seeds

Soybean (Glycine max) is a leguminous plant with a broad range of applications, particularly in agriculture and food production, where its seed composition—especially oil and protein content—is highly valued. Improving these traits is a primary focus of soybean breeding programs. In this study, we conducted a genome-wide association study (GWAS) to identify genetic loci linked to oil and protein content in seeds, using imputed genotype data for 180 Eurasian soybean varieties and the novel “genotypic twins” approach. This dataset encompassed 87 Russian and European cultivars and 93 breeding lines from Western Siberia. We identified 11 novel loci significantly associated with oil and protein content in seeds (p-value < 1.5 × 10−6), including one locus on chromosome 11 linked to protein content and 10 loci associated with oil content (chromosomes 1, 5, 11, 16, 17, and 18). The protein-associated locus is located near a gene encoding a CBL-interacting protein kinase, which is involved in key biological processes, including stress response mechanisms such as drought and osmotic stress. The oil-associated loci were linked to genes with diverse functions, including lipid transport, nutrient reservoir activity, and stress responses, such as Sec14p-like phosphatidylinositol transfer proteins and Germin-like proteins. These findings suggest that the loci identified not only influence oil and protein content but may also contribute to plant resilience under environmental stress conditions. The data obtained from this study provide valuable genetic markers that can be used in breeding programs to optimize oil and protein content, particularly in varieties adapted to Russian climates, and contribute to the development of high-yielding, nutritionally enhanced soybean cultivars.

Analysis of Phosphatidylinositol Transfer at ER-PM Contact Sites in Receptor-Stimulated Live Cells.

Phosphatidylinositol (PI) is an inositol-containing phospholipid synthesized in the endoplasmic reticulum (ER). PI is a precursor lipid for PI 4,5-bisphosphate (PI(4,5)P2) in the plasma membrane (PM) important for Ca2+ signaling in response to extracellular stimuli. Thus, ER-to-PM PI transfer becomes essential for cells to maintain PI(4,5)P2 homeostasis during receptor stimulation. In this chapter, we discuss two live-cell imaging protocols to analyze ER-to-PM PI transfer at ER-PM contact sites, where the two membrane compartments make close appositions accommodating PI transfer. First, we describe how to monitor PI(4,5)P2 replenishment following receptor stimulation as a readout of PI transfer using a PI(4,5)P2 biosensor and total internal reflection microscopy. The second protocol directly visualizes PI transfer proteins that accumulate at ER-PM contact sites and mediate PI(4,5)P2 replenishment with PI in the ER in stimulated cells. These methods provide spatial and temporal analysis of ER-to-PM PI transfer during receptor stimulation and can be adapted to other research questions related to this topic.

Genome-wide identification of Saccharum Sec14-like PITP gene family reveals that ScSEC14-1 is positively involved in disease resistance

Phosphatidylinositol transfer protein (PITP) plays an important role in lipid metabolism and plant immune response, however its function is less well-known in sugarcane (Saccharum spp.). In this study, 51 SsPITP genes clustered into four groups (I, III, IV, and V), were identified in S. spontaneum. Results showed that expansion of the SsPITP gene family was mainly through whole genome/segmental replication and dispersed replication events, while purification selection was the main driving force for its evolution. Besides, the SsPITP gene family contained cis-regulatory elements related to light, hormone, and stress response in promoter regions. Interestingly, SsPITPs were differentially expressed under Sporisorium scitamineum stress. Furthermore, due to the continuous high expression of SsPITP29 in the sugarcane resistant variety after being infected with S. scitamineum, its homologous gene ScSEC14–1 with 99.69 % amino acid identity was cloned from the sugarcane hybrid cultivar. The results of transient expression in tobacco epidermal cells showed that the ScSEC14–1 protein was localized on the cell membrane and nucleus. Notably, overexpression of ScSEC14–1 increased the expression of phospholipase C genes in transgenic plants by activating the phosphatidylinositol/calcium/MAPK signaling pathways and plant hormone signal transduction, increasing chlorophyll content and catalase activity, reducing malondialdehyde content, thus enhancing disease resistance. This work will help to systematically understand the characteristics of sugarcane Sec14-like PITP gene family and the disease resistance function of the ScSEC14–1.

PITPβ promotes COPI vesicle fission through lipid transfer and membrane contact formation.

Intracellular transport among organellar compartments occurs in two general ways, by membrane-bound carriers or membrane contacts. Specific circumstances that involve the coordination of these two modes of transport remain to be defined. Studying Coat Protein I (COPI) transport, we find that phosphatidylcholine with short acyl chains (sPC) is delivered through membrane contact from the endoplasmic reticulum (ER) to sites of COPI vesicle formation at the Golgi to support the fission stage. Phosphatidylinositol transfer protein beta (PITPβ) plays a key role in this process, with the elucidation of this role advancing a new understanding of how PITPβ acts, providing a mechanistic understanding of a specific circumstance when vesicular transport requires membrane contact, and contributing to a basic understanding of how transport carriers in a model intracellular pathway are formed.

Mammalian START-like phosphatidylinositol transfer proteins – Physiological perspectives and roles in cancer biology

PtdIns and its phosphorylated derivatives, the phosphoinositides, are the biochemical components of a major pathway of intracellular signaling in all eukaryotic cells. These lipids are few in terms of cohort of unique positional isomers, and are quantitatively minor species of the bulk cellular lipidome. Nevertheless, phosphoinositides regulate an impressively diverse set of biological processes. It is from that perspective that perturbations in phosphoinositide-dependent signaling pathways are increasingly being recognized as causal foundations of many human diseases – including cancer. Although phosphatidylinositol transfer proteins (PITPs) are not enzymes, these proteins are physiologically significant regulators of phosphoinositide signaling. As such, PITPs are conserved throughout the eukaryotic kingdom. Their biological importance notwithstanding, PITPs remain understudied. Herein, we review current information regarding PITP biology primarily focusing on how derangements in PITP function disrupt key signaling/developmental pathways and are associated with a growing list of pathologies in mammals.

Overexpression of PDR16 Confers Amphotericin B Resistance in a PMP3-Dependent Manner in Yeast Saccharomyces cerevisiae.

Invasive fungal infections in humans with compromised immune systems are the primary cause of morbidity and mortality, which is becoming more widely acknowledged. Amphotericin B (AmB) is one of the antifungal drugs used to treat such infections. AmB binds with plasma membrane ergosterol, inducing cellular ions to leak and causing cell death. Reduction in ergosterol content and modification of cell walls have been described as AmB resistance mechanisms. In addition, when the sphingolipid level is decreased, the cell becomes more susceptible to AmB. Previously, PDR16, a gene that encodes phosphatidylinositol transfer protein in Saccharomyces cerevisiae, was shown to enhance AmB resistance upon overexpression. However, the mechanism of PDR16-mediated AmB resistance is not clear. Here, in this study, it was discovered that a plasma membrane proteolipid 3 protein encoded by PMP3 is essential for PDR16-mediated AmB resistance. PDR16-mediated AmB resistance does not depend on ergosterol, but a functional sphingolipid biosynthetic pathway is required. Additionally, PMP3-mediated alteration in membrane integrity abolishes PDR16 mediated AmB resistance, confirming the importance of PMP3 in the PDR16 mediated AmB resistance.

Non-vesicular phosphatidylinositol transfer plays critical roles in defining organelle lipid composition

Phosphatidylinositol (PI) is the precursor lipid for the minor phosphoinositides (PPIns), which are critical for multiple functions in all eukaryotic cells. It is poorly understood how phosphatidylinositol, which is synthesized in the ER, reaches those membranes where PPIns are formed. Here, we used VT01454, a recently identified inhibitor of class I PI transfer proteins (PITPs), to unravel their roles in lipid metabolism, and solved the structure of inhibitor-bound PITPNA to gain insight into the mode of inhibition. We found that class I PITPs not only distribute PI for PPIns production in various organelles such as the plasma membrane (PM) and late endosomes/lysosomes, but that their inhibition also significantly reduced the levels of phosphatidylserine, di- and triacylglycerols, and other lipids, and caused prominent increases in phosphatidic acid. While VT01454 did not inhibit Golgi PI4P formation nor reduce resting PM PI(4,5)P2 levels, the recovery of the PM pool of PI(4,5)P2 after receptor-mediated hydrolysis required both class I and class II PITPs. Overall, these studies show that class I PITPs differentially regulate phosphoinositide pools and affect the overall cellular lipid landscape.

A genetic screen to uncover mechanisms underlying lipid transfer protein function at membrane contact sites.

Lipid transfer proteins mediate the transfer of lipids between organelle membranes, and the loss of function of these proteins has been linked to neurodegeneration. However, the mechanism by which loss of lipid transfer activity leads to neurodegeneration is not understood. In Drosophila photoreceptors, depletion of retinal degeneration B (RDGB), a phosphatidylinositol transfer protein, leads to defective phototransduction and retinal degeneration, but the mechanism by which loss of this activity leads to retinal degeneration is not understood. RDGB is localized to membrane contact sites through the interaction of its FFAT motif with the ER integral protein VAP. To identify regulators of RDGB function in vivo, we depleted more than 300 VAP-interacting proteins and identified a set of 52 suppressors of rdgB The molecular identity of these suppressors indicates a role of novel lipids in regulating RDGB function and of transcriptional and ubiquitination processes in mediating retinal degeneration in rdgB9 The human homologs of several of these molecules have been implicated in neurodevelopmental diseases underscoring the importance of VAP-mediated processes in these disorders.

Abstract 2365 Class I phosphatidylinositol transfer proteins control organelle membrane lipid composition beyond supporting phosphoinositides synthesis

Abstract 2365 Class I phosphatidylinositol transfer proteins control organelle membrane lipid composition beyond supporting phosphoinositides synthesis

A metabolically controlled contact site between vacuoles and lipid droplets in yeast

The lipid droplet (LD) organization proteins Ldo16 and Ldo45 affect multiple aspects of LD biology in yeast. They are linked to the LD biogenesis machinery seipin, and their loss causes defects in LD positioning, protein targeting, and breakdown. However, their molecular roles remained enigmatic. Here, we report that Ldo16/45 form a tether complex with Vac8 to create vacuole lipid droplet (vCLIP) contact sites, which can form in the absence of seipin. The phosphatidylinositol transfer protein (PITP) Pdr16 is a further vCLIP-resident recruited specifically by Ldo45. While only an LD subpopulation is engaged in vCLIPs at glucose-replete conditions, nutrient deprivation results in vCLIP expansion, and vCLIP defects impair lipophagy upon prolonged starvation. In summary, Ldo16/45 are multifunctional proteins that control the formation of a metabolically regulated contact site. Our studies suggest a link between LD biogenesis and breakdown and contribute to a deeper understanding of how lipid homeostasis is maintained during metabolic challenges.

PITPNC1 Suppress CD8+ T cell immune function and promote radioresistance in rectal cancer by modulating FASN/CD155

BackgroundRadioresistance is a primary factor contributing to the failure of rectal cancer treatment. Immune suppression plays a significant role in the development of radioresistance. We have investigated the potential role of phosphatidylinositol transfer protein cytoplasmic 1 (PITPNC1) in regulating immune suppression associated with radioresistance.MethodsTo elucidate the mechanisms by which PITPNC1 influences radioresistance, we established HT29, SW480, and MC38 radioresistant cell lines. The relationship between radioresistance and changes in the proportion of immune cells was verified through subcutaneous tumor models and flow cytometry. Changes in the expression levels of PITPNC1, FASN, and CD155 were determined using immunohistochemistry and western blotting techniques. The interplay between these proteins was investigated using immunofluorescence co-localization and immunoprecipitation assays. Additionally, siRNA and lentivirus-mediated gene knockdown or overexpression, as well as co-culture of tumor cells with PBMCs or CD8+ T cells and establishment of stable transgenic cell lines in vivo, were employed to validate the impact of the PITPNC1/FASN/CD155 pathway on CD8+ T cell immune function.ResultsUnder irradiation, the apoptosis rate and expression of apoptosis-related proteins in radioresistant colorectal cancer cell lines were significantly decreased, while the cell proliferation rate increased. In radioresistant tumor-bearing mice, the proportion of CD8+ T cells and IFN-γ production within immune cells decreased. Immunohistochemical analysis of human and animal tissue specimens resistant to radiotherapy showed a significant increase in the expression levels of PITPNC1, FASN, and CD155. Gene knockdown and rescue experiments demonstrated that PITPNC1 can regulate the expression of CD155 on the surface of tumor cells through FASN. In addition, co-culture experiments and in vivo tumor-bearing experiments have shown that silencing PITPNC1 can inhibit FASN/CD155, enhance CD8+ T cell immune function, promote colorectal cancer cell death, and ultimately reduce radioresistance in tumor-bearing models.ConclusionsPITPNC1 regulates the expression of CD155 through FASN, inhibits CD8+ T cell immune function, and promotes radioresistance in rectal cancer.

The Vacuole Lipid Droplet Contact Site vCLIP

Lipid droplets frequently form contact sites with the membrane of the vacuole, the lysosome-like organelle in yeast. These vacuole lipid droplet (vCLIP) contact sites respond strongly to metabolic cues: while only a subset of lipid droplets is bound to the vacuole when nutrients are abundant, other metabolic states induce stronger contact site formation. Physical lipid droplet-vacuole binding is related to the process of lipophagy, a lipid droplet-specific form of microautophagy. The molecular basis for the formation and function of vCLIP contact sites remained enigmatic for a long time. This knowledge gap was filled when it was found that vCLIP is formed by the structurally related lipid droplet tether proteins Ldo16 and Ldo45, and the vacuolar surface protein Vac8. Ldo45 additionally recruits the phosphatidylinositol transfer protein Pdr16 to vCLIP. Here, we review the literature on the lipid droplet-vacuole contact site in light of the progress in our understanding of its molecular basis and discuss future directions for the field.

Prognostic role and therapeutic implications of phosphatidylinositol transfer protein cytoplasmic 1 in primary prostate cancer

Prognostic role and therapeutic implications of phosphatidylinositol transfer protein cytoplasmic 1 in primary prostate cancer

Microcolin H, a novel autophagy inducer, exerts potent antitumour activity by targeting PITPα/β

The identification of effective drug targets and the development of bioactive molecules are areas of high need in cancer therapy. The phosphatidylinositol transfer protein alpha/beta isoform (PITPα/β) has been reported to play an essential role in integrating phosphoinositide trafficking and lipid metabolism in diverse cellular processes but remains unexplored as a potential target for cancer treatment. Herein, data analysis of clinical cancer samples revealed that PITPα/β expression is closely correlated with the poor prognosis. Target identification by chemical proteomic methods revealed that microcolin H, a naturally occurring marine lipopeptide, directly binds PITPα/β and displays antiproliferative activity on different types of tumour cell lines. Furthermore, we identified that microcolin H treatment increased the conversion of LC3I to LC3II, accompanied by a reduction of the level of p62 in cancer cells, leading to autophagic cell death. Moreover, microcolin H showed preeminent antitumour efficacy in nude mouse subcutaneous tumour models with low toxicity. Our discoveries revealed that by targeting PITPα/β, microcolin H induced autophagic cell death in tumours with efficient anti-proliferating activity, which sheds light on PITPα/β as a promising therapeutic target for cancer treatment.

The expanding roles of PI4P and PI(4,5)P2 at the plasma membrane: Role of phosphatidylinositol transfer proteins

Phosphoinositides are phosphorylated derivatives of phosphatidylinositol, a phospholipid that is synthesised at the endoplasmic reticulum. The plasma membrane contains the enzymes to phosphorylate phosphatidylinositol and is therefore rich in the phosphorylated derivatives, PI4P and PI(4,5)P2. PI(4,5)P2 is a substrate for phospholipase C and during cell signaling, PI(4,5)P2 levels are reduced. Here I discuss a family of proteins, phosphatidylinositol transfer proteins (PITPs) that can restore PI(4,5)P2 levels.

Synthesis of a photocleavable bola-phosphatidylcholine

Phosphatidylinositol transfer proteins (PITPs) are ubiquitous in eukaryotes and are involved in the regulation of phospholipid metabolism, membrane trafficking, and signal transduction. Sec14 is a yeast PITP that has been shown to transfer phosphatidylinositol (PI) or phosphatidylcholine (PC) from the endoplasmic reticulum to the Golgi. It is now believed that Sec14 may play a greater role than just shuttling PI and PC throughout the cell. Genetic evidence suggests that retrieval of membrane-bound PI by Sec14 also manages to present PI to the phosphatidylinositol-4-kinase, Pik1, to generate phosphatidylinositol-4-phosphate, PI(4)P. To test this hypothetical model, we designed a photocleavable bolalipid to span the entire membrane, having one phosphatidylcholine or phosphatidylinositol headgroup on each leaflet connected by a photocleavable diacid. Sec14 should not be able to present the bola-PI to Pik1 for phosphorylation as the head group will be difficult to lift from the bilayer as it is tethered on the opposite leaflet. After photocleavage the two halves would behave as a normal phospholipid, thus phosphorylation by Pik1 would resume. We report here the synthesis of a photocleavable bola-PC, a precursor to the desired bola-PI. The mono-photocleavable bola-PC lipid was designed to contain two glycerol molecules with choline head groups connected through a phosphodiester bond at the sn3 position. Each glycerol was acylated with palmitic acid at the sn1 position. These two glycerol moieties were then connected through their respective sn2 hydroxyls via a photocleavable dicarboxylic acid containing a nitrophenyl ethyl photolabile protecting group. The bola-PC and its precursors were found to undergo efficient photocleavage when irradiated in solution or in vesicles with 365 nm light for two minutes. Treatment of the bola-PC with a mutant phospholipase D and myo-inositol produced a mono-inositol bola-PC-PI.

Rescuing β cells.

Loss of phosphatidylinositol transfer protein alpha underlies pancreatic β cell failure.

Restoration of PITPNA in Type 2 diabetic human islets reverses pancreatic beta-cell dysfunction

Defects in insulin processing and granule maturation are linked to pancreatic beta-cell failure during type 2 diabetes (T2D). Phosphatidylinositol transfer protein alpha (PITPNA) stimulates activity of phosphatidylinositol (PtdIns) 4-OH kinase to produce sufficient PtdIns-4-phosphate (PtdIns-4-P) in the trans-Golgi network to promote insulin granule maturation. PITPNA in beta-cells of T2D human subjects is markedly reduced suggesting its depletion accompanies beta-cell dysfunction. Conditional deletion of Pitpna in the beta-cells of Ins-Cre, Pitpnaflox/flox mice leads to hyperglycemia resulting from decreasing glucose-stimulated insulin secretion (GSIS) and reducing pancreatic beta-cell mass. Furthermore, PITPNA silencing in human islets confirms its role in PtdIns-4-P synthesis and leads to impaired insulin granule maturation and docking, GSIS, and proinsulin processing with evidence of ER stress. Restoration of PITPNA in islets of T2D human subjects reverses these beta-cell defects and identify PITPNA as a critical target linked to beta-cell failure in T2D.

The lipid transfer proteins Nir2 and Nir3 sustain phosphoinositide signaling and actin dynamics during phagocytosis.

Changes in membrane phosphoinositides and local Ca2+ elevations at sites of particle capture coordinate the dynamic remodeling of the actin cytoskeleton during phagocytosis. Here, we show that the phosphatidylinositol (PI) transfer proteins PITPNM1 (Nir2) and PITPNM2 (Nir3) maintain phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] homeostasis at phagocytic cups, thereby promoting actin contractility and the sealing of phagosomes. Nir3 and to a lesser extent Nir2 accumulated on endoplasmic reticulum (ER) cisternae juxtaposed to phagocytic cups when expressed in phagocytic COS-7 cells. CRISPR-Cas9 editing of Nir2 and Nir3 genes decreased plasma membrane PI(4,5)P2 levels, store-operated Ca2+ entry (SOCE) and receptor-mediated phagocytosis, stalling particle capture at the cup stage. Re-expression of either Nir2 or Nir3 restored phagocytosis, but not SOCE, proportionally to the PM PI(4,5)P2 levels. Phagosomes forming in Nir2 and Nir3 (Nir2/3) double-knockout cells had decreased overall PI(4,5)P2 levels but normal periphagosomal Ca2+ signals. Nir2/3 depletion reduced the density of contractile actin rings at sites of particle capture, causing repetitive low-intensity contractile events indicative of abortive phagosome closure. We conclude that Nir proteins maintain phosphoinositide homeostasis at phagocytic cups, thereby sustaining the signals that initiate the remodeling of the actin cytoskeleton during phagocytosis.

Role of SEC14-like phosphatidylinositol transfer proteins in membrane identity and dynamics.

Membrane identity and dynamic processes, that act at membrane sites, provide important cues for regulating transport, signal transduction and communication across membranes. There are still numerous open questions as to how membrane identity changes and the dynamic processes acting at the surface of membranes are regulated in diverse eukaryotes in particular plants and which roles are being played by protein interaction complexes composed of peripheral and integral membrane proteins. One class of peripheral membrane proteins conserved across eukaryotes comprises the SEC14-like phosphatidylinositol transfer proteins (SEC14L-PITPs). These proteins share a SEC14 domain that contributes to membrane identity and fulfills regulatory functions in membrane trafficking by its ability to sense, bind, transport and exchange lipophilic substances between membranes, such as phosphoinositides and diverse other lipophilic substances. SEC14L-PITPs can occur as single-domain SEC14-only proteins in all investigated organisms or with a modular domain structure as multi-domain proteins in animals and streptophytes (comprising charales and land plants). Here, we present an overview on the functional roles of SEC14L-PITPs, with a special focus on the multi-domain SEC14L-PITPs of the SEC14-nodulin and SEC14-GOLD group (PATELLINs, PATLs in plants). This indicates that SEC14L-PITPs play diverse roles from membrane trafficking to organism fitness in plants. We concentrate on the structure of SEC14L-PITPs, their ability to not only bind phospholipids but also other lipophilic ligands, and their ability to regulate complex cellular responses through interacting with proteins at membrane sites.