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Phospholipase A 2 's (PLA 2 's) constitute a superfamily of enzymes that hydrolyze the sn -2 fatty acyl chain on glycerophospholipids. We have previously reported that each PLA 2 Type shows a unique substrate specificity for the molecular species it hydrolyzes, especially the acyl chain that is cleaved from the sn -2 position and to some extent the polar group. However, phosphatidylinositol (PI) and PI phosphates (PIPs) have not been as well studied as substrates as other phospholipids because the PIPs require adaptation of the standard analysis methods, but they are important in vivo. We determined the in vitro activity of the three major types of human PLA 2 's, namely the cytosolic (c), calcium-independent (i), and secreted (s) PLA 2 's toward PI, PI-4-phosphate (PI(4)P), and PI-4,5-bisphosphate (PI(4,5)P 2). The in vitro assay revealed that Group IVA cPLA 2 (GIVA cPLA 2) showed relatively high activity toward PI and PI(4)P among the tested PLA 2 's; nevertheless, the highly hydrophilic headgroup disrupted the interaction between the lipid surface and the enzyme. GIVA cPLA 2 and GVIA iPLA 2 showed detectable activity toward PI(4,5)P 2 , but it appeared to be a poorer substrate for all of the PLA 2 's tested. Furthermore, molecular dynamics (MD) simulations demonstrated that Thr416 and Glu418 of GIVA cPLA 2 contribute significantly to accommodating the hydrophilic head groups of PI and PI(4)P, which could explain some selectivity for PI and PI(4)P. These results indicated that GIVA cPLA 2 can accommodate PI and PI(4)P in its active site and hydrolyze them, suggesting that the GIVA cPLA 2 may best account for the PI and PIP hydrolysis in living cells. • GIVA cPLA 2 showed significant activity toward PI and PI(4)P in neutralized micelles. • GIVA cPLA 2 showed minimal activity toward PI(4,5)P2. • GVIA-2 iPLA 2 showed less activity toward PI, PI(4)P and PI(4,5)P2 than GIVA cPLA 2. • GV sPLA 2 showed minimal activity toward PI and none toward PI(4)P and PI(4,5)P2. • Thr416 and Glu418 in GIVA cPLA 2 play critical roles in accepting the PI head group. [ABSTRACT FROM AUTHOR]

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. 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. [ABSTRACT FROM AUTHOR]

Circular actin waves that propagate on the substrate-attached membrane of Dictyostelium cells separate two distinct membrane domains from each other: an inner territory rich in phosphatidyl-(3,4,5) trisphosphate (PIP3) and an external area decorated with the PIP3-degrading 3-phosphatase PTEN. During wave propagation, the inner territory increases at the expense of the external area. Beyond a size limit, the inner territory becomes unstable, breaking into an inner and an external domain. The sharp boundary between these domains is demarcated by the insertion of an actin wave. During the conversion of inner territory to external area, the state of the membrane fluctuates, as visualized by dynamic landscapes of formin B binding. Here we analyze the formin B fluctuations in relation to three markers of the membrane state: activated Ras, PIP3, and PTEN., Competing Interests: Declaration of interests The authors declare no competing or financial interests., (Copyright © 2023 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Background: The transformation of the supported lipid bilayer (SLB) membrane by extracted cytosol from living resources, has recently drawn much attention. It enables us to address the question of whether the purified phospholipid SLB membrane, including lipids related to amoeba locomotion, which was discussed in many previous studies, exhibits membrane deformation in the presence of cytosol extracted from amoeba; Methods: In this report, a method for reconstituting a supported lipid bilayer (SLB) membrane, composed of purified phospholipids and cytosol extracted from Dictyostelium discoideum, is described. This technique is a new reconstitution method combining the artificial constitution of membranes with the reconstitution using animate cytosol (without precise purification at a molecular level), contributing to membrane deformation analysis; Results: The morphology transition of a SLB membrane composed of phosphatidylcholines, after the addition of cytosolic extract, was traced using a confocal laser scanning fluorescence microscope. As a result, pore formation in the SLB membrane was observed and phosphatidylinositides incorporated into the SLB membrane tended to suppress pore formation and expansion; Conclusions: The current findings imply that phosphatidylinositides have the potential to control cytoplasm activity and bind to a phosphoinositide-containing SLB membrane. [ABSTRACT FROM AUTHOR]

Background: The cytosol of amoeba cells controls the membrane deformation during their motion in vivo. To investigate such ability of the cytosol of amoeba cell, Dictyostelium discoideum (Dictyostelium), in vitro, we used lipids extracted from Dictyostelium and commercially available phospholipids, and prepared substrate-supported lipid membrane patches on the micrometer scale by spin coating. Results: We found that the spin coater holder, which has pores (pore size = 3.1 mm) of negative pressure to hold the cover glass induced the concave surface of the cover glass. The membrane lipid patches were formed at each position in the vicinity of the holder pores and their sizes were in the range of 2.7 to 3.2 × 104 μm². After addition of the cytosol extracted from Dictyostelium to the lipid membrane patches, through time-lapse observation with a confocal laser scanning fluorescence microscope, we observed an autonomous buckling of the Dictyostelium lipid patches and localized behaviours of proteins found within. Conclusion: The current method serves as the novel technique for the preparation of film patches in which the positions of patches are controlled by the holder pores without fabricating, modifying, and arranging the chemical properties of the solution components of lipids. The findings imply that lipid-binding proteins in the cytosol were adsorbed and accumulated within the Dictyostelium lipid patches, inducing the transformation of the cell-sized patch. [ABSTRACT FROM AUTHOR]

Abstract: Phosphatidylinositides, most negatively charged lipids in cellular membranes, regulate diverse effector proteins through the interaction with their lipid binding domains. We have previously reported inhibitory effect of small chemicals on the interaction between PtdIns(3,4,5)P3 and Btk PH domain. Here, we report that the inhibitory effects of same sets of chemicals on Grp1 PH domain and epsin1 ENTH domain to elucidate diversity of inhibitory mechanisms upon different lipid binding domains. Among the chemicals, chemical 8 showed best inhibition in vitro assay for Grp1 PH domain and epsin1 ENTH domain, and then the interaction between small chemicals and lipid binding domains was further investigated by in silico docking experiments. As a result, it was concluded that the diverse inhibitory effects on different lipid binding domains were dependent on not only the number of interactions between small chemical and domain, but also additional interaction with positively charged surfaces as the secondary binding sites. This finding will help to develop lipid binding inhibitors as antagonists for lipid–protein interactions, and these inhibitors would be novel therapeutic drug candidates via regulating effector proteins involved in severe human diseases. [Copyright &y& Elsevier]

Abstract: The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated Cl− channel critically important in Cl− secreting epithelia. Mutations in the CFTR gene, such as ΔF508CFTR leads to cystic fibrosis, a severe disease with defective Cl− secretion. CFTR is stimulated by the serum and glucocorticoid-inducible kinase SGK1. The SGK1 dependent regulation of several carriers and channels involves the phosphatidylinositol-3-phosphate-5-kinase PIKfyve, which similarly mediates the regulation of glucose carriers by PKB/Akt. The present study was thus performed to elucidate whether PKB/Akt and PIKfyve are regulators of CFTR. To this end CFTR or ΔF508CFTR were expressed in Xenopus oocytes alone or together with PKB, PIKfyve or the SGK1/PKB resistant mutant S318APIKfyve, and the current generated by cAMP upregulation with 10μM forskolin+1mM IBMX determined utilizing dual electrode voltage clamp. As a result, forskolin/IBMX treatment triggered a current (I cAMP) in CFTR-expressing Xenopus oocytes, but not in oocytes expressing ΔF508CFTR. Coexpression of PKB/Akt and PIKfyve, but not of S318APIKfyve, stimulated I cAMP in CFTR-expressing (≈2- to 3-fold) but not in ΔF508CFTR-expressing or water injected Xenopus oocytes. Immunohistochemistry revealed that the coexpression of PIKfyve, but not of S318APIKfyve, enhanced the CFTR protein abundance but not the ΔF508CFTR protein abundance in CFTR or ΔF508CFTR-expressing oocytes. The present observations reveal a novel powerful regulator of intact but not of defective CFTR. [Copyright &y& Elsevier]

The two mammalian phosphatidylcholine (PC)-selective phospholipase D (PLD) enzymes remove the choline head group from PC to produce phosphatidic acid (PA). PA stimulates phosphatidylinositol(4)phosphate 5-kinases, can function as a binding site for membrane proteins, is required for certain membrane fusion or fission events and is an important precursor for the production of diacylglycerol (DAG). Both PA and DAG are lipids that favor negatively curved membranes rather than planar bilayers and can reduce the energetic barrier to membrane fission and fusion. Recent data provide a mechanistic explanation for the role PLDs play in some aspects of membrane traffic and provide an explanation for why some membrane fusion reactions require PA and some do not. PLDs also act as guanosine triphosphatase-activating proteins for dynamin and may participate with dynamin in the process of vesicle fission. [ABSTRACT FROM AUTHOR]

The assembly of stable cytoskeletal structures from dynamically recycled molecules requires developmental and spatial regulation of protein interactions. In muscle, titin acts as a molecular ruler organizing the actin cytoskeleton via interactions with many sarcomeric proteins, including the crosslinking protein α-actinin. An interaction between the C-terminal domain of α-actinin and titin Z-repeat motifs targets α-actinin to the Z-disk. Here we investigate the cellular regulation of this interaction. α-actinin is a rod shaped head-to-tail homodimer. In contrast to C-terminal fragments, full-length α-actinin does not bind Z-repeats. We identify a 30-residue Z-repeat homologous sequence between the actin-binding and rod regions of α-actinin that binds the C-terminal domain with nanomolar affinity. Thus, Z-repeat binding is prevented by this ‘pseudoligand’ interaction between the subunits of the α-actinin dimer. This autoinhibition is relieved upon binding of the Z-disk lipid phosphatidylinositol-bisphosphate to the actin-binding domain. We suggest that this novel mechanism is relevant to control the site-specific interactions of α-actinin during sarcomere assembly and turnover. The intramolecular contacts defined here also constrain a structural model for intrasterical regulation of all α-actinin isoforms. [ABSTRACT FROM AUTHOR]

Abstract: In previous studies we found that μ-opioids, acting via μ-opioid receptors, inhibit endothelin-stimulated C6 glioma cell growth. In the preceding article we show that the κ-selective opioid agonist U69,593 acts as a mitogen with a potency similar to that of endothelin in the same astrocytic model system. Here we report that C6 cell treatment with μ-opioid agonists for 1 h results in the inhibition of κ-opioid mitogenic signaling. The μ-selective agonist endomorphin-1 attenuates κ-opioid-stimulated DNA synthesis, phosphoinositide turnover, and extracellular signal-regulated kinase phosphorylation. To investigate the role of receptor endocytosis in signaling, we have examined the effects of dynamin-1 and its GTPase-defective, dominant suppressor mutant (K44A) on opioid modulation of extracellular signal-regulated kinase phosphorylation in C6 cells. Overexpression of dynamin K44A in C6 cells does not affect κ-opioid phosphorylation of extracellular signal-regulated kinase. However, it does block the inhibitory action on κ-opioid signaling mediated by the κ-opioid receptor. Our results are consistent with a growing body of evidence of the opposing actions of μ- and κ-opioids and provide new insight into the role of opioid receptor trafficking in signaling. [ABSTRACT FROM AUTHOR]

Addition of purified plasmin or plasminogen (0.1 μ M) to serum-free culture media elevated cellular D- myo-inositol 1,4,5-trisphosphate (Ins P) levels in human colorectal carcinoma cells within 1 h to double those of control cells. This was accompanied by decreases in cellular phosphatidylinositol bisphosphate by 40% in cells exposed to fibrinolytic ligands for up to 1 h. The effect was not due to opening of Ca channels of the type blocked by 5 μ M nifedipine, and 100 μ M EGTA, a Ca chelator, did not suppress plasmin's ability to elevate Ins P. Binding assays at 4° C withI-labelled plasmin indicated maximum binding within 1 h suggesting that the effects of plasmin may be associated with its cell-binding function. These cells could convert exogenous plasminogen to plasmin with endogenous activation and this was accompanied by a decrease in radioactive phosphatidylinositol well below control levels (13% of control). Our results contribute to evidence for the association of plasmin-binding sites with a signalling system. A cell signalling system indirectly or directly associated with plasmin binding, would permit carcinoma cells to coordinate extracellular fibrinolysis with cell migration and motility through second messengers. [ABSTRACT FROM AUTHOR]