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Journal of Bioenergetics and... Aug 2024Lysophosphatidic acid (LPA) is a simple lipid which is endogenously synthesized from lysophosphatidylcholine (LPC) by autotaxin (ATX). LPA mediates a variety of cellular... (Review)
Review
Lysophosphatidic acid (LPA) is a simple lipid which is endogenously synthesized from lysophosphatidylcholine (LPC) by autotaxin (ATX). LPA mediates a variety of cellular responses through the binding of G protein-coupled LPA receptors (LPA to LPA). It is considered that LPA receptor-mediated signaling plays an important role in the pathogenesis of human malignancy. Genetic alterations and epigenetic changes of LPA receptors have been detected in some cancer cells as well as LPA per se. Moreover, LPA receptors contribute to the promotion of tumor progression, including cell proliferation, invasion, metastasis, tumorigenicity, and angiogenesis. In recent studies, the activation of LPA receptor-mediated signaling regulates chemoresistance and radiosensitivity in cancer cells. This review provides an updated overview on the roles of LPA receptor-mediated signaling in the regulation of cancer cell functions and its potential utility as a molecular target for novel therapies in clinical cancer approaches.
Topics: Humans; Receptors, Lysophosphatidic Acid; Neoplasms; Signal Transduction; Lysophospholipids; Animals
PubMed: 38886303
DOI: 10.1007/s10863-024-10028-9 -
Archives of Biochemistry and Biophysics Jun 2024Formation of transport vesicles requires the coordinate activity of the coating machinery that selects cargo into the nascent vesicle and the membrane bending machinery...
Formation of transport vesicles requires the coordinate activity of the coating machinery that selects cargo into the nascent vesicle and the membrane bending machinery that imparts curvature to the forming bud. Vesicle coating at the trans-Golgi Network (TGN) involves AP1, GGA2 and clathrin, which are recruited to membranes by activated ARF GTPases. The ARF activation at the TGN is mediated by the BIG1 and BIG2 guanine nucleotide exchange factors (GEFs). Membrane deformation at the TGN has been shown to be mediated by lipid flippases, including ATP8A1, that moves phospholipids from the inner to the outer leaflet of the TGN membrane. We probed a possible coupling between the coating and deformation machineries by testing for an interaction between BIG1, BIG2 and ATP8A1, and by assessing whether such an interaction may influence coating efficiency. Herein, we document that BIG1 and BIG2 co-localize with ATP8A1 in both, static and highly mobile TGN elements, and that BIG1 and BIG2 bind ATP8A1. We show that the interaction involves the catalytic Sec7 domain of the GEFs and the cytosolic C-terminal tail of ATP8A1. Moreover, we report that the expression of ATP8A1, but not ATP8A1 lacking the GEF-binding cytosolic tail, increases the generation of activated ARFs at the TGN and increases the selective recruitment of AP1, GGA2 and clathrin to TGN membranes. This occurs without increasing BIG1 or BIG2 levels at the TGN, suggesting that the binding of the ATP8A1 flippase tail to the Sec7 domain of BIG1/BIG2 increases their catalytic activity. Our results support a model in which a flippase component of the deformation machinery impacts the activity of the GEF component of the coating machinery.
PubMed: 38879142
DOI: 10.1016/j.abb.2024.110049 -
Cellular and Molecular Life Sciences :... Jun 2024Blood ultrafiltration in nephrons critically depends on specialized intercellular junctions between podocytes, named slit diaphragms (SDs). Here, by studying a...
Blood ultrafiltration in nephrons critically depends on specialized intercellular junctions between podocytes, named slit diaphragms (SDs). Here, by studying a homologous structure found in Drosophila nephrocytes, we identify the phospholipid scramblase Scramb1 as an essential component of the SD, uncovering a novel link between membrane dynamics and SD formation. In scramb1 mutants, SDs fail to form. Instead, the SD components Sticks and stones/nephrin, Polychaetoid/ZO-1, and the Src-kinase Src64B/Fyn associate in cortical foci lacking the key SD protein Dumbfounded/NEPH1. Scramb1 interaction with Polychaetoid/ZO-1 and Flotillin2, the presence of essential putative palmitoylation sites and its capacity to oligomerize, suggest a function in promoting SD assembly within lipid raft microdomains. Furthermore, Scramb1 interactors as well as its functional sensitivity to temperature, suggest an active involvement in membrane remodeling processes during SD assembly. Remarkably, putative Ca-binding sites in Scramb1 are essential for its activity raising the possibility that Ca signaling may control the assembly of SDs by impacting on Scramb1 activity.
Topics: Animals; Podocytes; Drosophila Proteins; Phospholipid Transfer Proteins; Membrane Proteins; Drosophila melanogaster; Membrane Microdomains; Intercellular Junctions
PubMed: 38878170
DOI: 10.1007/s00018-024-05287-z -
Genetics Jun 2024To survive daily damage, the formation of actomyosin ring at the wound edge is required to rapidly close cell wounds. Calcium influx is one of the start signals for...
To survive daily damage, the formation of actomyosin ring at the wound edge is required to rapidly close cell wounds. Calcium influx is one of the start signals for these cell wound repair events. Here, we find that rapid recruitment of all three Drosophila calcium responding and phospholipid binding Annexin proteins (AnxB9, AnxB10, AnxB11) to distinct regions around the wound is regulated by the quantity of calcium influx rather than their binding to specific phospholipids. The distinct recruitment patterns of these Annexins regulate the subsequent recruitment of RhoGEF2 and RhoGEF3 through actin stabilization to form a robust actomyosin ring. Surprisingly, while the wound does not close in the absence of calcium influx, we find that reduced calcium influx can still initiate repair processes, albeit leading to severe repair phenotypes. Thus, our results suggest that, in addition to initiating repair events, the quantity of calcium influx is important for precise Annexin spatiotemporal protein recruitment to cell wounds and efficient wound repair.
PubMed: 38874345
DOI: 10.1093/genetics/iyae101 -
Biotechnology and Bioengineering Jun 2024The demand for Lentiviral Vector (LV) drug substance is increasing. However, primary capture using convective anion-exchange chromatography remains a significant...
The demand for Lentiviral Vector (LV) drug substance is increasing. However, primary capture using convective anion-exchange chromatography remains a significant manufacturing challenge. This stems from a poor understanding of the complex adsorption behaviors linked to LVs intricate and variable structure, such as high binding heterogeneity which is typically characterized by a gradient elution profile consisting of two peaks. Understanding which LV structural components drive these phenomena is therefore crucial for rational process design. This work identifies the key LV envelope components responsible for binding to quaternary-amine membrane adsorbents. Eliminating the pseudotype protein (Vesicular Stomatitis Virus G glycoprotein [VSV-G]) did not impact the heterogenous two-peak elution profile, suggesting it is not a major binding species. Digestion of envelope glycosaminoglycans (GAGs), present on proteoglycans, leads to a dramatic reduction in the proportion of vector eluted in peak 2, decreasing from 50% to 3.1%, and a threefold increase in peak 1 maximum. Data from reinjection experiments point towards interparticle envelope heterogeneity from discrete LV populations, where the two-peak profile emerges from a subpopulation of LVs interacting via highly charged GAGs (peak 2) along with a weaker binding population likely interacting through the phospholipid membrane and envelope protein (peak 1).
PubMed: 38874319
DOI: 10.1002/bit.28766 -
Nature Communications Jun 2024Focal adhesions form liquid-like assemblies around activated integrin receptors at the plasma membrane. How they achieve their flexible properties is not well...
Focal adhesions form liquid-like assemblies around activated integrin receptors at the plasma membrane. How they achieve their flexible properties is not well understood. Here, we use recombinant focal adhesion proteins to reconstitute the core structural machinery in vitro. We observe liquid-liquid phase separation of the core focal adhesion proteins talin and vinculin for a spectrum of conditions and interaction partners. Intriguingly, we show that binding to PI(4,5)P-containing membranes triggers phase separation of these proteins on the membrane surface, which in turn induces the enrichment of integrin in the clusters. We suggest a mechanism by which 2-dimensional biomolecular condensates assemble on membranes from soluble proteins in the cytoplasm: lipid-binding triggers protein activation and thus, liquid-liquid phase separation of these membrane-bound proteins. This could explain how early focal adhesions maintain a structured and force-resistant organization into the cytoplasm, while still being highly dynamic and able to quickly assemble and disassemble.
Topics: Talin; Focal Adhesions; Cell Membrane; Vinculin; Humans; Animals; Phosphatidylinositol 4,5-Diphosphate; Integrins; Cytoplasm; Protein Binding; Phase Separation
PubMed: 38862544
DOI: 10.1038/s41467-024-49222-z -
Plant Physiology and Biochemistry : PPB Jun 2024The enzyme phospholipase A (PLA) plays a crucial role in acyl remodeling of phospholipids via the Lands' cycle, and consequently alters fatty acid compositions in...
The enzyme phospholipase A (PLA) plays a crucial role in acyl remodeling of phospholipids via the Lands' cycle, and consequently alters fatty acid compositions in triacylglycerol (TAG). In this study, a full-length cDNA sequence coding Myrmecia incisa phospholipase A (MiPLA) was cloned using the technique of rapid amplification of cDNA ends. Comparison of the 1082-bp cDNA with its corresponding cloned DNA sequence revealed that MiPLA contained 3 introns. Mature MiPLA (mMiPLA) had a conserved Ca-binding loop and a catalytic site motif that has been recognized in plant secretory PLA (sPLA) proteins. Correspondingly, phylogenetic analysis illustrated that MiPLA was clustered within GroupXIA of plant sPLA proteins. To ascertain the function of MiPLA, the cDNA coding for mMiPLA was subcloned into the vector pET-32a to facilitate the production of recombinant mMiPLA in Escherichia coli. Recombinant mMiPLA was purified and used for the in vitro enzyme reaction. Thin-layer chromatography profiles of the catalytic products generated by recombinant mMiPLA indicated a specificity for cleaving sn-2 acyl chains from phospholipids, thereby functionally characterizing MiPLA. Although recombinant mMiPLA displayed a strong preference for phosphatidylethanolamine, it preferentially hydrolyzes arachidonic acid (ArA) at the sn-2 position of phosphatidylcholine. Results from the fused expression of p1300-sp-EGFP-mMiPLA illustrated that MiPLA was localized in the intercellular space of onion epidermis. Furthermore, the positive correlation between MiPLA transcription and free ArA levels were established. Consequently, the role of mMiPLA in the biosynthesis of ArA-rich TAG was elucidated. This study helps to understand how M. incisa preferentially uses ArA to synthesize TAG.
PubMed: 38861822
DOI: 10.1016/j.plaphy.2024.108806 -
Journal of Biochemistry Jun 2024Chondroitin sulfate (CS) is a linear polysaccharide chain of alternating residues of glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc), modified with sulfate...
Chondroitin sulfate (CS) is a linear polysaccharide chain of alternating residues of glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc), modified with sulfate groups. Based on the structure, CS chains bind to bioactive molecules specifically and regulate their functions. For example, CS whose GalNAc is sulfated at the C4 position, termed CSA, and CS whose GalNAc is sulfated at both C4 and C6 positions, termed CSE, bind to a malaria protein VAR2CSA and receptor type of protein tyrosine phosphatase sigma (RPTPσ), respectively in a specific manner. Here, we modified CSA and CSE chains with phosphatidylethanolamine (PE) at a reducing end, attached them to liposomes containing phospholipids, and generated CSA- and CSE-liposomes. The CS-PE was incorporated into the liposome particles efficiently. Inhibition ELISA revealed specific interaction of CSA and CSE with recombinant VAR2CSA and RPTPσ, respectively, more efficiently than CS chains alone. Furthermore, CSE-liposome was specifically incorporated into RPTPσ-expressing HEK293T cells. These results indicate CS-liposome as a novel and efficient drug delivery system, especially for CS-binding molecules.
PubMed: 38861406
DOI: 10.1093/jb/mvae041 -
Analytical Chemistry Jun 2024Lipids are critical modulators of membrane protein structure and function. However, it is challenging to investigate the thermodynamics of protein-lipid interactions...
Lipids are critical modulators of membrane protein structure and function. However, it is challenging to investigate the thermodynamics of protein-lipid interactions because lipids can simultaneously bind membrane proteins at different sites with different specificities. Here, we developed a native mass spectrometry (MS) approach using single and double mutants to measure the relative energetic contributions of specific residues on Aquaporin Z (AqpZ) toward cardiolipin (CL) binding. We first mutated potential lipid-binding residues on AqpZ, and mixed mutant and wild-type proteins together with CL. By using native MS to simultaneously resolve lipid binding to the mutant and wild-type proteins in a single spectrum, we directly determined the relative affinities of CL binding, thereby revealing the relative Gibbs free energy change for lipid binding caused by the mutation. Comparing different mutants revealed that W14 contributes to the tightest CL binding site, with R224 contributing to a lower affinity site. Using double mutant cycling, we investigated the synergy between W14 and R224 sites on CL binding. Overall, this novel native MS approach provides unique insights into the binding of lipids to specific sites on membrane proteins.
Topics: Cardiolipins; Mass Spectrometry; Mutation; Aquaporins; Binding Sites; Protein Binding; Membrane Proteins; Thermodynamics; Models, Molecular; Escherichia coli Proteins
PubMed: 38859611
DOI: 10.1021/acs.analchem.4c01704 -
BMC Genomics Jun 2024Phospholipases constitute a diverse category of enzymes responsible for the breakdown of phospholipids. Their involvement in signal transduction with a pivotal role in...
BACKGROUND
Phospholipases constitute a diverse category of enzymes responsible for the breakdown of phospholipids. Their involvement in signal transduction with a pivotal role in plant development and stress responses is well documented.
RESULTS
In the present investigation, a thorough genome-wide analysis revealed that the pearl millet genome contains at least 44 phospholipase genes distributed across its 7 chromosomes, with chromosome one harbouring the highest number of these genes. The synteny analysis suggested a close genetic relationship of pearl millet phospholipases with that of foxtail millet and sorghum. All identified genes were examined to unravel their gene structures, protein attributes, cis-regulatory elements, and expression patterns in two pearl millet genotypes contrasting for rancidity. All the phospholipases have a high alpha-helix content and distorted regions within the predicted secondary structures. Moreover, many of these enzymes possess binding sites for both metal and non-metal ligands. Additionally, the putative promoter regions associated with these genes exhibit multiple copies of cis-elements specifically responsive to biotic and abiotic stress factors and signaling molecules. The transcriptional profiling of 44 phospholipase genes in two genotypes contrasting for rancidity across six key tissues during pearl millet growth revealed a predominant expression in grains, followed by seed coat and endosperm. Specifically, the genes PgPLD-alpha1-1, PgPLD-alpha1-5, PgPLD-delta1-7a, PgPLA1-II-1a, and PgPLD-delta1-2a exhibited notable expression in grains of both the genotypes while showing negligible expression in the other five tissues. The sequence alignment of putative promoters revealed several variations including SNPs and InDels. These variations resulted in modifications to the corresponding cis-acting elements, forming distinct transcription factor binding sites suggesting the transcriptional-level regulation for these five genes in pearl millet.
CONCLUSIONS
The current study utilized a genome-wide computational analysis to characterize the phospholipase gene family in pearl millet. A comprehensive expression profile of 44 phospholipases led to the identification of five grain-specific candidates. This underscores a potential role for at least these five genes in grain quality traits including the regulation of rancidity in pearl millet. Therefore, this study marks the first exploration highlighting the possible impact of phospholipases towards enhancing agronomic traits in pearl millet.
Topics: Pennisetum; Phospholipases; Multigene Family; Edible Grain; Gene Expression Regulation, Plant; Promoter Regions, Genetic; Phylogeny; Plant Proteins; Synteny; Gene Expression Profiling; Genotype; Chromosome Mapping
PubMed: 38858648
DOI: 10.1186/s12864-024-10504-x