Search Results (40)

The Phosphoinositide Specific-Phospholipase C (PI-PLC) family of enzymes plays a crucial role in various cellular processes by catalyzing the hydrolysis of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] into inositol 1,4,5-trisphosphate (IP₃) and diacylglycerol (DAG), which are essential messengers mediating critical intracellular signaling pathways. Herein, we carry out a comprehensive analysis of the structure, function, regulation, and implications of the PI-PLC family enzymes in both physiological and pathological contexts. More specifically, we discuss the structural features of PI-PLCs, elucidating their conserved domains and catalytic mechanisms. Furthermore, we explore the multifaceted roles of PI-PLCs in signal transduction, cellular homeostasis, and membrane dynamics, whilst highlighting the intricate regulatory mechanisms governing their activity such as protein–protein interactions, post-translational modifications, and lipid modulation. Lastly, we assess the involvement of PI-PLCs in various diseases, such as cancer, neurological disorders, immune dysregulation, and male infertility, emphasizing their potential as therapeutic targets. Full article

This study investigates the effect of dietary Lactobacillus plantarum supplementation on juvenile coho salmon (Oncorhynchus kisutch). Four groups of the juveniles (initial weight 103.87 ± 2.65 g) were fed for 10 weeks with four diets containing 0 (control diet), 10⁵ (T1), 10⁷ (T2), and 10⁹ (T3) cfu/g of L. plantarum. The main results are as follows: Compared with the control diet, the final weight, specific growth rate (SGR), and weight gain rate (WGR) of the juveniles fed the T1, T2, and T3 diet significantly (p < 0.05) increased, while the feed coefficient ratio (FCR) expressed an opposite trend. The activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-PX) in the serum of the juveniles fed the T2 diet significantly (p < 0.05) increased, while the malondialdehyde (MDA) expressed an opposite trend. The expression of phosphatidylinositol 4,5-bisphosphate 3-kinase (pi3k), AKT-interacting protein (akt), mechanistic target of rapamycin kinase (mtor), glucose-6-phosphate dehydrogenase (g6pd), sod, cat, and gsh-px genes in the liver of the juveniles fed the T2 diet significantly (p < 0.05) increased. In conclusion, the T2 diet significantly improved the growth performance, antioxidant capacity, and upregulated key mTOR pathway genes in juvenile coho salmon. Full article

Enveloped viruses rely on host membranes for trafficking and assembly. A substantial body of literature published over the years supports the involvement of cellular membrane lipids in the enveloped virus assembly processes. In particular, the knowledge regarding the relationship between viral structural proteins and acidic phospholipids has been steadily increasing in recent years. In this review, we will briefly review the cellular functions of plasma membrane-associated acidic phospholipids and the mechanisms that regulate their local distribution within this membrane. We will then explore the interplay between viruses and the plasma membrane acidic phospholipids in the context of the assembly process for two enveloped viruses, the influenza A virus (IAV) and the human immunodeficiency virus type 1 (HIV-1). Among the proteins encoded by these viruses, three viral structural proteins, IAV hemagglutinin (HA), IAV matrix protein-1 (M1), and HIV-1 Gag protein, are known to interact with acidic phospholipids, phosphatidylserine and/or phosphatidylinositol (4,5)-bisphosphate. These interactions regulate the localization of the viral proteins to and/or within the plasma membrane and likely facilitate the clustering of the proteins. On the other hand, these viral proteins, via their ability to multimerize, can also alter the distribution of the lipids and may induce acidic-lipid-enriched membrane domains. We will discuss the potential significance of these interactions in the virus assembly process and the property of the progeny virions. Finally, we will outline key outstanding questions that need to be answered for a better understanding of the relationships between enveloped virus assembly and acidic phospholipids. Full article

Retroviral assembly is a highly coordinated step in the replication cycle. The process is initiated when the newly synthesized Gag and Gag-Pol polyproteins are directed to the inner leaflet of the plasma membrane (PM), where they facilitate the budding and release of immature viral particles. Extensive research over the years has provided crucial insights into the molecular determinants of this assembly step. It is established that Gag targeting and binding to the PM is mediated by interactions of the matrix (MA) domain and acidic phospholipids such as phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂). This binding event, along with binding to viral RNA, initiates oligomerization of Gag on the PM, a process mediated by the capsid (CA) domain. Much of the previous studies have focused on human immunodeficiency virus type 1 (HIV-1). Although the general steps of retroviral replication are consistent across different retroviruses, comparative studies revealed notable differences in the structure and function of viral components. In this review, we present recent findings on the assembly mechanisms of Human T-cell leukemia virus type 1 and highlight key differences from HIV-1, focusing particularly on the molecular determinants of Gag–PM interactions and CA assembly. Full article

Melatonin plays a critical role in regulating embryo attachment in ruminants. While numerous studies have investigated its effects on early embryo development in vitro, the precise mechanisms by which melatonin influences the receptivity of endometrial epithelial cells in dairy cows remain unclear. The prerequisite for embryo implantation is the specific physiological condition of the endometrium that allows the embryo to implant, also known as endometrial receptivity. In addition to this, endometrial cells undergo processes such as proliferation, differentiation, and renewal, which makes the embryo more easily implanted. In this study, bovine endometrial epithelial cells were cultured and treated with melatonin, Silent Information Regulator 1 (SIRT1) inhibitor (EX527), and protein kinase B (AKT) phosphorylation inhibitor (periposine). RT-qPCR, Western blot, and immunofluorescence analysis were performed to investigate the effects of melatonin on the expression of target gene (SIRT1); cell proliferative genes, phosphatidylinositol-4,5-bisphosphate 3-Kinase (PI3K), AKT, cyclinD1, cyclinE1; and receptive genes (Leukemia Inhibitory Factor (LIF), Vascular Endothelial Growth Factor (VEGF), Homeobox Structure Gene 10 (HOXA10)). Additionally, microRNA (miRNA) mimics and inhibitors were used to transfect the cells to study the regulatory relationship between miRNA and receptive genes. Results indicated that melatonin activates the PI3K/AKT signaling pathway, upregulates cyclinD1 and cyclinE1, and promotes the proliferation of bovine endometrial epithelial cells. Melatonin also upregulated the expression of VEGF and HOXA10 and downregulated the expression of bta-miR-497 and bta-miR-27a-3p through SIRT1/PI3K/AKT signaling pathway. Further, bta-miR-497 and bta-miR-27a-3p were found to negatively regulate VEGF and HOXA10, respectively. Therefore, melatonin regulates the expression of VEGF and HOXA10 through the SIRT1/PI3K/AKT pathway and promotes the establishment of receptivity in bovine endometrial epithelial cells. Full article

Lamins, the nuclear intermediate filaments, are important regulators of nuclear structural integrity as well as nuclear functional processes such as DNA transcription, replication and repair, and epigenetic regulations. A portion of phosphorylated lamin A/C localizes to the nuclear interior in interphase, forming a lamin A/C pool with specific properties and distinct functions. Nucleoplasmic lamin A/C molecular functions are mainly dependent on its binding partners; therefore, revealing new interactions could give us new clues on the lamin A/C mechanism of action. In the present study, we show that lamin A/C interacts with nuclear phosphoinositides (PIPs), and with nuclear myosin I (NM1). Both NM1 and nuclear PIPs have been previously reported as important regulators of gene expression and DNA damage/repair. Furthermore, phosphorylated lamin A/C forms a complex with NM1 in a phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2)-dependent manner in the nuclear interior. Taken together, our study reveals a previously unidentified interaction between phosphorylated lamin A/C, NM1, and PI(4,5)P2 and suggests new possible ways of nucleoplasmic lamin A/C regulation, function, and importance for the formation of functional nuclear microdomains. Full article

Murine leukemia viruses (MuLVs) are simple retroviruses that cause several diseases in mice. Retroviruses encode three basic genes: gag, pol, and env. Gag is translated as a polyprotein and moves to assembly sites where viral particles are shaped by cleavage of poly-Gag. Viral release depends on the intracellular trafficking of viral proteins, which is determined by both viral and cellular factors. ADP-ribosylation factor 6 (Arf6) is a small GTPase that regulates vesicular trafficking and recycling of different types of cargo in cells. Arf6 also activates phospholipase D (PLD) and phosphatidylinositol-4-phosphate 5-kinase (PIP5K) and produces phosphatidylinositol-4,5-bisphosphate (PI(4,5)P₂). We investigated how Arf6 affected MuLV release with a constitutively active form of Arf6, Arf6Q67L. Expression of Arf6Q67L impaired Gag release by accumulating Gag at PI(4,5)P₂-enriched compartments in the cytoplasm. Treatment of the inhibitors for PLD and PIP5K impaired or recovered MuLV Gag release in the cells expressing GFP (control) and Arf6Q67L, implying that regulation of PI(4,5)P₂ through PLD and PIP5K affected MuLV release. Interference with the phosphoinositide 3-kinases, mammalian target of rapamycin (mTOR) pathway, and vacuolar-type ATPase activities showed further impairment of Gag release from the cells expressing Arf6Q67L. In contrast, mTOR inhibition increased Gag release in the control cells. The proteasome inhibitors reduced viral release in the cells regardless of Arf6Q67L expression. These data outline the differences in MuLV release under the controlled and overactivated Arf6 conditions and provide new insight into pathways for MuLV release. Full article

The type I phosphatidylinositol 4-phosphate 5-kinase (PIP5K) family produces the critical lipid regulator phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) in the plasma membrane (PM). Here, we investigated the potential role of PIP5Kγ, a PIP5K isoform, in the Hippo pathway. The ectopic expression of PIP5Kγ87 or PIP5Kγ90, two major PIP5Kγ splice variants, activated large tumor suppressor kinase 1 (LATS1) and inhibited Yes-associated protein (YAP), whereas PIP5Kγ knockdown yielded opposite effects. The regulatory effects of PIP5Kγ were dependent on its catalytic activity and the presence of Merlin and LATS1. PIP5Kγ knockdown weakened the restoration of YAP phosphorylation upon stimulation with epidermal growth factor or lysophosphatidic acid. We further found that PIP5Kγ90 bound to the Merlin’s band 4.1/ezrin/radixin/moesin (FERM) domain, forming a complex with PI(4,5)P2 and LATS1 at the PM. Notably, PIP5Kγ90, but not its kinase-deficient mutant, potentiated Merlin–LATS1 interaction and recruited LATS1 to the PM. Consistently, PIP5Kγ knockdown or inhibitor (UNC3230) enhanced colony formation in carcinoma cell lines YAP-dependently. In addition, PIP5Kγ90 interacted with heat shock cognate 71-kDa protein (Hsc70), which also contributed to Hippo pathway activation. Collectively, our results suggest that PIP5Kγ regulates the Hippo–YAP pathway by forming a functional complex with Merlin and LATS1 at the PI(4,5)P2-rich PM and via interplay with Hsc70. Full article

PIK3R1 (also known as p85α) is a regulatory subunit of phosphoinositide 3-kinases (PI3Ks). PI3K, a heterodimer of a regulatory subunit and a catalytic subunit, phosphorylates phosphatidylinositol into secondary signaling molecules involved in regulating metabolic homeostasis. PI3K converts phosphatidylinositol 4,5-bisphosphate (PIP₂) to phosphatidylinositol 3,4,5-triphosphate (PIP₃), which recruits protein kinase AKT to the inner leaflet of the cell membrane to be activated and to participate in various metabolic functions. PIK3R1 stabilizes and inhibits p110 catalytic activity and serves as an adaptor to interact with insulin receptor substrate (IRS) proteins and growth factor receptors. Thus, mutations in PIK3R1 or altered expression of PIK3R1 could modulate the activity of PI3K and result in significant metabolic outcomes. Interestingly, recent studies also found PI3K-independent functions of PIK3R1. Overall, in this article, we will provide an updated review of the metabolic functions of PIK3R1 that includes studies of PIK3R1 in various metabolic tissues using animal models, the mechanisms modulating PIK3R1 activity, and studies on the mutations of human PIK3R1 gene. Full article

Phosphoinositides (PIs) are small, phosphorylated lipids that serve many functions in the cell. They regulate endo- and exocytosis, vesicular trafficking, actin reorganization, and cell mobility, and they act as signaling molecules. The most abundant PIs in the cell are phosphatidylinositol-4-monophosphate (PI4P) and phosphatidylinositol-4,5-bisphosphate [PI(4,5)P₂]. PI4P is mostly localized at the Golgi apparatus where it regulates the anterograde trafficking from the Golgi apparatus to the plasma membrane (PM), but it also localizes at the PM. On the other hand, the main localization site of PI(4,5)P₂ is the PM where it regulates the formation of endocytic vesicles. The levels of PIs are regulated by many kinases and phosphatases. Four main kinases phosphorylate the precursor molecule phosphatidylinositol into PI4P, divided into two classes (PI4KIIα, PI4KIIβ, PI4KIIIα, and PI4KIIIβ), and three main kinases phosphorylate PI4P to form PI(4,5)P₂ (PI4P5KIα, PI4P5KIβ, and PI4P5KIγ). In this review, we discuss the localization and function of the kinases that produce PI4P and PI(4,5)P₂, as well as the localization and function of their product molecules with an overview of tools for the detection of these PIs. Full article

The HIV-1 Gag protein binds to the host cell membrane and assembles into immature particles. Then, in the course of immature virion budding, activated protease cleaves Gag into its main components: MA, CA, NC, and p6 proteins. The highly basic residues of MA predominantly interact with the acidic head of phosphatidyl-inositol-4,5-bisphosphate (PI(4,5)P2) inserted into the membrane. Our research group developed L-Heptanoylphosphatidyl Inositol Pentakisphosphate (L-HIPPO) and previously confirmed that this compound bound to the MA more strongly than PI(4,5)P2 and inositol hexakisphosphate (IP6) did. Therefore, herein we rationally designed eight new L-HIPPO derivatives based on the fact that the most changeable parts of L-HIPPO were two acyl chains. After that, we employed molecular docking for eight compounds via Maestro software using high-resolution crystal structures of MA in complex with IP6 (PDB IDs: 7E1I, 7E1J, and 7E1K), which were recently elucidated by our research group. The most promising docking scores were obtained with benzene-inserted compounds. Thus, we generated a library containing 213 new aromatic group-inserted L-HIPPO derivatives and performed the same molecular docking procedure. According to the results, we determined the nine new L-HIPPO derivatives most effectively binding to the MA with the most favorable scoring functions and pharmacokinetic properties for further exploration. Full article

Despite its low abundance, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) is a key modulator of membrane-associated signaling events in eukaryotic cells. Temporal and spatial regulation of PI(4,5)P₂ concentration can achieve localized increases in the levels of this lipid, which are crucial for the activation or recruitment of peripheral proteins to the plasma membrane. The recent observation of the dramatic impact of physiological divalent cation concentrations on PI(4,5)P₂ clustering, suggests that protein anchoring to the plasma membrane through PI(4,5)P₂ is likely not defined solely by a simple (monomeric PI(4,5)P₂)/(protein bound PI(4,5)P₂) equilibrium, but instead depends on complex protein interactions with PI(4,5)P₂ clusters. The insertion of PI(4,5)P₂-binding proteins within these clusters can putatively modulate protein–protein interactions in the membrane, but the relevance of such effects is largely unknown. In this work, we characterized the impact of Ca²⁺ on the organization and protein–protein interactions of PI(4,5)P₂-binding proteins. We show that, in giant unilamellar vesicles presenting PI(4,5)P₂, the membrane diffusion properties of pleckstrin homology (PH) domains tagged with a yellow fluorescent protein (YFP) are affected by the presence of Ca²⁺, suggesting direct interactions between the protein and PI(4,5)P₂ clusters. Importantly, PH-YFP is found to dimerize in the membrane in the absence of Ca²⁺. This oligomerization is inhibited in the presence of physiological concentrations of the divalent cation. These results confirm that cation-dependent PI(4,5)P₂ clustering promotes interactions between PI(4,5)P₂-binding proteins and has the potential to dramatically influence the organization and downstream interactions of PI(4,5)P₂-binding proteins in the plasma membrane. Full article

Two-pore channels (TPCs) are ligand-gated cation-selective ion channels that are preserved in plant and animal cells. In the latter, TPCs are located in membranes of acidic organelles, such as endosomes, lysosomes, and endolysosomes. Here, we focus on the function of these unique ion channels in mast cells, which are leukocytes that mature from myeloid hematopoietic stem cells. The cytoplasm of these innate immune cells contains a large number of granules that comprise messenger substances, such as histamine and heparin. Mast cells, along with basophil granulocytes, play an essential role in anaphylaxis and allergic reactions by releasing inflammatory mediators. Signaling in mast cells is mainly regulated via the release of Ca²⁺ from the endoplasmic reticulum as well as from acidic compartments, such as endolysosomes. For the crosstalk of these organelles TPCs seem essential. Allergic reactions and anaphylaxis were previously shown to be associated with the endolysosomal two-pore channel TPC1. The release of histamine, controlled by intracellular Ca²⁺ signals, was increased upon genetic or pharmacologic TPC1 inhibition. Conversely, stimulation of TPC channel activity by one of its endogenous ligands, namely nicotinic adenine dinucleotide phosphate (NAADP) or phosphatidylinositol 3,5-bisphosphate (PI(3,5)P₂), were found to trigger the release of Ca²⁺ from the endolysosomes; thereby improving the effect of TPC1 on regulated mast cell degranulation. In this review we discuss the importance of TPC1 for regulating Ca²⁺ homeostasis in mast cells and the overall potential of TPC1 as a pharmacological target in anti-inflammatory therapy. Full article

HIV-1 viral particle assembly occurs specifically at the plasma membrane and is driven primarily by the viral polyprotein Gag. Selective association of Gag with the plasma membrane is a key step in the viral assembly pathway, which is traditionally attributed to the MA domain. MA regulates specific plasma membrane binding through two primary mechanisms including: (1) specific interaction of the MA highly basic region (HBR) with the plasma membrane phospholipid phosphatidylinositol (4,5) bisphosphate [PI(4,5)P₂], and (2) tRNA binding to the MA HBR, which prevents Gag association with non-PI(4,5)P₂ containing membranes. Gag multimerization, driven by both CA–CA inter-protein interactions and NC-RNA binding, also plays an essential role in viral particle assembly, mediating the establishment and growth of the immature Gag lattice on the plasma membrane. In addition to these functions, the multimerization of HIV-1 Gag has also been demonstrated to enhance its membrane binding activity through the MA domain. This review provides an overview of the mechanisms regulating Gag membrane binding through the MA domain and multimerization through the CA and NC domains, and examines how these two functions are intertwined, allowing for multimerization mediated enhancement of Gag membrane binding. Full article

Defensins form an integral part of the cationic host defence peptide (HDP) family, a key component of innate immunity. Apart from their antimicrobial and immunomodulatory activities, many HDPs exert multifaceted effects on tumour cells, notably direct oncolysis and/or inhibition of tumour cell migration. Therefore, HDPs have been explored as promising anticancer therapeutics. Human β-defensin 2 (HBD-2) represents a prominent member of human HDPs, being well-characterised for its potent pathogen-killing, wound-healing, cytokine-inducing and leukocyte-chemoattracting functions. However, its anticancer effects remain largely unknown. Recently, we demonstrated that HBD-2 binds strongly to phosphatidylinositol-4,5-bisphosphate (PI(4,5)P₂), a key mediator of defensin-induced cell death and an instructional messenger during cell migration. Hence, in this study, we sought to investigate the lytic and anti-migratory effects of HBD-2 on tumour cells. Using various cell biological assays and confocal microscopy, we showed that HBD-2 killed tumour cells via acute lytic cell death rather than apoptosis. In addition, our data suggested that, despite the reported PI(4,5)P₂ interaction, HBD-2 does not affect cytoskeletal-dependent tumour cell migration. Together, our findings provide further insights into defensin biology and informs future defensin-based drug development. Full article