-
Molecular Cell Jun 2022Phospholipase A2, group VII (PLA2G7) is widely recognized as a secreted, lipoprotein-associated PLA2 in plasma that converts phospholipid platelet-activating factor...
Phospholipase A2, group VII (PLA2G7) is widely recognized as a secreted, lipoprotein-associated PLA2 in plasma that converts phospholipid platelet-activating factor (PAF) to a biologically inactive product Lyso-PAF during inflammatory response. We report that intracellular PLA2G7 is selectively important for cell proliferation and tumor growth potential of melanoma cells expressing mutant NRAS, but not cells expressing BRAF V600E. Mechanistically, PLA2G7 signals through its product Lyso-PAF to contribute to RAF1 activation by mutant NRAS, which is bypassed by BRAF V600E. Intracellular Lyso-PAF promotes p21-activated kinase 2 (PAK2) activation by binding to its catalytic domain and altering ATP kinetics, while PAK2 significantly contributes to S338-phosphorylation of RAF1 in addition to PAK1. Furthermore, the PLA2G7-PAK2 axis is also required for full activation of RAF1 in cells stimulated by epidermal growth factor (EGF) or cancer cells expressing mutant KRAS. Thus, PLA2G7 and Lyso-PAF exhibit intracellular signaling functions as key elements of RAS-RAF1 signaling.
Topics: Phospholipases A2; Phospholipids; Platelet Activating Factor; Proto-Oncogene Proteins B-raf
PubMed: 35417664
DOI: 10.1016/j.molcel.2022.03.026 -
Biomolecules Aug 2023Oxidative stress is a well-known hallmark of Antiphospholipid Antibody Syndrome (APS), a systemic autoimmune disease characterized by arterial and venous thrombosis... (Review)
Review
Oxidative stress is a well-known hallmark of Antiphospholipid Antibody Syndrome (APS), a systemic autoimmune disease characterized by arterial and venous thrombosis and/or pregnancy morbidity. Oxidative stress may affect various signaling pathways and biological processes, promoting dysfunctional immune responses and inflammation, inducing apoptosis, deregulating autophagy and impairing mitochondrial function. The chronic oxidative stress and the dysregulation of the immune system leads to the loss of tolerance, which drives autoantibody production and inflammation with the development of endothelial dysfunction. In particular, anti-phospholipid antibodies (aPL), which target phospholipids and/or phospholipid binding proteins, mainly β-glycoprotein I (β-GPI), play a functional role in the cell signal transduction pathway(s), thus contributing to oxidative stress and thrombotic events. An oxidation-antioxidant imbalance may be detected in the blood of patients with APS as a reflection of disease progression. This review focuses on functional evidence highlighting the role of oxidative stress in the initiation and progression of APS. The protective role of food supplements and Nuclear Factor Erythroid 2-Related Factor 2 (NRF2) activators in APS patients will be summarized to point out the potential of these therapeutic approaches to reduce APS-related clinical complications.
Topics: Female; Pregnancy; Humans; Antibodies, Antiphospholipid; NF-E2-Related Factor 2; Oxidative Stress; Phospholipids; Antioxidants
PubMed: 37627286
DOI: 10.3390/biom13081221 -
Nature Communications Oct 2023Cryptococcus spp. are environmental fungi that first must adapt to the host environment before they can cause life-threatening meningitis in immunocompromised patients....
Cryptococcus spp. are environmental fungi that first must adapt to the host environment before they can cause life-threatening meningitis in immunocompromised patients. Host CO concentrations are 100-fold higher than the external environment and strains unable to grow at host CO concentrations are not pathogenic. Using a genetic screening and transcriptional profiling approach, we report that the TOR pathway is critical for C. neoformans adaptation to host CO partly through Ypk1-dependent remodeling of phosphatidylserine asymmetry at the plasma membrane. We also describe a C. neoformans ABC/PDR transporter (PDR9) that is highly expressed in CO-sensitive environmental strains, suppresses CO-induced phosphatidylserine/phospholipid remodeling, and increases susceptibility to host concentrations of CO. Interestingly, regulation of plasma membrane lipid asymmetry by the TOR-Ypk1 axis is distinct in C. neoformans compared to S. cerevisiae. Finally, host CO concentrations suppress the C. neoformans pathways that respond to host temperature (Mpk1) and pH (Rim101), indicating that host adaptation requires a stringent balance among distinct stress responses.
Topics: Humans; Cryptococcus neoformans; Saccharomyces cerevisiae; Phospholipids; Carbon Dioxide; Phosphatidylserines; Cryptococcosis; ATP-Binding Cassette Transporters
PubMed: 37852972
DOI: 10.1038/s41467-023-42318-y -
Nature Communications Sep 2023The "eat me" signal, phosphatidylserine is exposed on the surface of dying cells by phospholipid scrambling. Previously, we showed that the Xkr family protein Xkr4 is...
The "eat me" signal, phosphatidylserine is exposed on the surface of dying cells by phospholipid scrambling. Previously, we showed that the Xkr family protein Xkr4 is activated by caspase-mediated cleavage and binding of the XRCC4 fragment. Here, we show that extracellular calcium is an additional factor needed to activate Xkr4. The constitutively active mutant of Xkr4 is found to induce phospholipid scrambling in an extracellular, but not intracellular, calcium-dependent manner. Importantly, other Xkr family members also require extracellular calcium for activation. Alanine scanning shows that D123 and D127 of TM1 and E310 of TM3 coordinate calcium binding. Moreover, lysine scanning demonstrates that the E310K mutation-mediated salt bridge between TM1 and TM3 bypasses the requirement of calcium. Cysteine scanning proves that disulfide bond formation between TM1 and TM3 also activates phospholipid scrambling without calcium. Collectively, this study shows that extracellular calcium functions as a molecular glue for TM1 and TM3 of Xkr proteins for activation, thus demonstrating a regulatory mechanism for multi-transmembrane region-containing proteins.
Topics: Calcium; Alanine; Biological Transport; Caspases; Phosphatidylserines
PubMed: 37696806
DOI: 10.1038/s41467-023-40934-2 -
Biochemistry Jun 2020Activation of G-protein-gated inwardly rectifying potassium channels (Kir3.) requires the direct binding of phosphorylated phosphatidylinositides (PIPs). Previous...
Activation of G-protein-gated inwardly rectifying potassium channels (Kir3.) requires the direct binding of phosphorylated phosphatidylinositides (PIPs). Previous studies have established that PIP isoforms activate Kir channels to varying degrees and the binding affinity between PIPs and Kir3.2 appears to be correlated with the level of activation. However, how individual residues contribute to the selectivity of Kir channels toward PIP isoforms is poorly understood. Here, we employ native mass spectrometry (MS) and fluorescent lipid binding assays to gain insight into the contribution of specific Kir3.2 residues binding to phospholipids. For the wild-type channel, we demonstrate the importance of membrane protein samples devoid of co-purified contaminants for protein-lipid binding studies and show that PIP(4,5)P cooperatively binds Kir3.2 with a Hill coefficient of 2.7. We also find lipid binding profiles determined from native MS and solution binding assays are in direct agreement. Point mutations of Kir3.2 residues that interact with PIPs distinctly alter selective lipid binding. The K64Q mutation results in altered binding profiles with the highest binding affinity for PIP(4,5)P with specific acyl chains. Mutation of R92 to Pro, a residue found in Kir6.2, results in promiscuous binding of PIP isoforms. Kir3.2 with the K194A mutation results in a distinct binding preference for PIP(3,4,5)P over other PIP isoforms. Taken together, our results underscore the utmost importance of protein quality for protein-lipid binding studies and show that a single mutation in Kir3.2 can alter the selectivity toward PIPs.
Topics: Animals; Fluorescence Resonance Energy Transfer; G Protein-Coupled Inwardly-Rectifying Potassium Channels; Mass Spectrometry; Mice; Phosphatidylinositols; Point Mutation
PubMed: 32372643
DOI: 10.1021/acs.biochem.0c00163 -
Biochimica Et Biophysica Acta.... Oct 2023In phagocytes, superoxide anion (O), the precursor of reactive oxygen species, is produced by the NADPH oxidase complex to kill pathogens. Phagocyte NADPH oxidase...
In phagocytes, superoxide anion (O), the precursor of reactive oxygen species, is produced by the NADPH oxidase complex to kill pathogens. Phagocyte NADPH oxidase consists of the transmembrane cytochrome b (cyt b) and four cytosolic components: p40, p47, p67, and Rac1/2. The phagocyte activation by stimuli leads to activation of signal transduction pathways. This is followed by the translocation of cytosolic components to the membrane and their association with cyt b to form the active enzyme. To investigate the roles of membrane-interacting domains of the cytosolic proteins in the NADPH oxidase complex assembly and activity, we used giant unilamellar phospholipid vesicles (GUV). We also used the neutrophil-like cell line PLB-985 to investigate these roles under physiological conditions. We confirmed that the isolated proteins must be activated to bind to the membrane. We showed that their membrane binding was strengthened by the presence of the other cytosolic partners, with a key role for p47. We also used a fused chimera consisting of p47(aa 1-286), p67(aa 1-212) and Rac1Q61L, as well as mutated versions in the p47 PX domain and the Rac polybasic region (PB). We showed that these two domains have a crucial role in the trimera membrane-binding and in the trimera assembly to cyt b. They also have an impact on O.- production in vitro and in cellulo: the PX domain strongly binding to GUV made of a mix of polar lipids; and the PB region strongly binding to the plasma membrane of neutrophils and resting PLB-985 cells.
Topics: Phospholipids; Cytochromes b; Phagocytes; NADPH Oxidases; Cell Membrane; Binding Sites
PubMed: 37245861
DOI: 10.1016/j.bbamem.2023.184180 -
Cells May 2021Signal transduction, the ability of cells to perceive information from the surroundings and alter behavior in response, is an essential property of life. Studies on... (Review)
Review
Signal transduction, the ability of cells to perceive information from the surroundings and alter behavior in response, is an essential property of life. Studies on tyrosine kinase action fundamentally changed our concept of cellular regulation. The induced assembly of subcellular hubs via the recognition of local protein or lipid modifications by modular protein interactions is now a central paradigm in signaling. Such molecular interactions are mediated by specific protein interaction domains. The first such domain identified was the SH2 domain, which was postulated to be a reader capable of finding and binding protein partners displaying phosphorylated tyrosine side chains. The SH3 domain was found to be involved in the formation of stable protein sub-complexes by constitutively attaching to proline-rich surfaces on its binding partners. The SH2 and SH3 domains have thus served as the prototypes for a diverse collection of interaction domains that recognize not only proteins but also lipids, nucleic acids, and small molecules. It has also been found that particular SH2 and SH3 domains themselves might also bind to and rely on lipids to modulate complex assembly. Some lipid-binding properties of SH2 and SH3 domains are reviewed here.
Topics: Animals; Binding Sites; Humans; Phospholipids; Phosphorylation; Protein Binding; Protein Interaction Domains and Motifs; Signal Transduction; Structure-Activity Relationship; src Homology Domains; src-Family Kinases
PubMed: 34068055
DOI: 10.3390/cells10051191 -
Journal of Integrative Plant Biology Jan 2021The plant cytoskeleton undergoes dynamic remodeling in response to diverse developmental and environmental cues. Remodeling of the cytoskeleton coordinates growth in... (Review)
Review
The plant cytoskeleton undergoes dynamic remodeling in response to diverse developmental and environmental cues. Remodeling of the cytoskeleton coordinates growth in plant cells, including trafficking and exocytosis of membrane and wall components during cell expansion, and regulation of hypocotyl elongation in response to light. Cytoskeletal remodeling also has key functions in disease resistance and abiotic stress responses. Many stimuli result in altered activity of cytoskeleton-associated proteins, microtubule-associated proteins (MAPs) and actin-binding proteins (ABPs). MAPs and ABPs are the main players determining the spatiotemporally dynamic nature of the cytoskeleton, functioning in a sensory hub that decodes signals to modulate plant cytoskeletal behavior. Moreover, MAP and ABP activities and levels are precisely regulated during development and environmental responses, but our understanding of this process remains limited. In this review, we summarize the evidence linking multiple signaling pathways, MAP and ABP activities and levels, and cytoskeletal rearrangements in plant cells. We highlight advances in elucidating the multiple mechanisms that regulate MAP and ABP activities and levels, including calcium and calmodulin signaling, ROP GTPase activity, phospholipid signaling, and post-translational modifications.
Topics: Cytoskeleton; Microfilament Proteins; Microtubule-Associated Proteins; Phospholipids; Protein Processing, Post-Translational; Signal Transduction
PubMed: 33274838
DOI: 10.1111/jipb.13046 -
Biochimica Et Biophysica Acta.... Feb 2022Annexin A2 (AnxA2) is a calcium- and phospholipid-binding protein that plays roles in cellular processes involving membrane and cytoskeleton dynamics and is able to...
Annexin A2 (AnxA2) is a calcium- and phospholipid-binding protein that plays roles in cellular processes involving membrane and cytoskeleton dynamics and is able to associate to several partner proteins. However, the principal molecular partners of AnxA2 are negatively charged phospholipids such as phosphatidylserine and phosphatidyl-inositol-(4,5)-phosphate. Herein we have studied different aspects of membrane lipid rearrangements induced by AnxA2 membrane binding. X-ray diffraction data revealed that AnxA2 has the property to stabilize lamellar structures and to block the formation of highly curved lipid phases (inverted hexagonal phase, H). By using pyrene-labelled cholesterol and the environmental probe di-4-ANEPPDHQ, we observed that in model membranes, AnxA2 is able to modify both, cholesterol distribution and lipid compaction. In epithelial cells, we observed that AnxA2 localizes to membranes of different lipid order. The protein binding to membranes resulted in both, increases and/or decreases in membrane order depending on the cellular membrane regions. Overall, AnxA2 showed the capacity to modulate plasma membrane properties by inducing lipid redistribution that may lead to an increase in order or disorder of the membranes.
Topics: Annexin A2; Biophysical Phenomena; Calcium; Carrier Proteins; Cell Communication; Cell Membrane; Cytoskeleton; Humans; Membrane Lipids; Phosphatidylserines; Phospholipids
PubMed: 34699769
DOI: 10.1016/j.bbamem.2021.183810 -
Metabolism: Clinical and Experimental Aug 2023Homeostasis of autophagy under normal conditions and nutrient stress is maintained by adaptive activation of regulatory proteins. However, the protein-lipid crosstalk...
BACKGROUND
Homeostasis of autophagy under normal conditions and nutrient stress is maintained by adaptive activation of regulatory proteins. However, the protein-lipid crosstalk that modulates the switch from suppression to activation of autophagy initiation is largely unknown.
RESULTS
Here, we show that human diazepam-binding inhibitor (DBI), also known as acyl-CoA binding protein (ACBP), binds to phosphatidylethanolamine of the phagophore membrane under nutrient-rich growth conditions, leading to inhibition of LC3 lipidation and suppression of autophagy initiation. Specific residues, including the conserved tyrosine residues of DBI, interact with phosphatidylethanolamine to stabilize the later molecule in the acyl-CoA binding cavity of the protein. Under starvation, phosphorylation of serine-21 of DBI mediated by the AMP-activated protein kinase results in a drastic reduction in the affinity of the protein for phosphatidylethanolamine. The release of serine-21 phosphorylated DBI from the phagophore upon nutrient starvation restores the high LC3 lipidation flux and maturation of the phagophore to autophagosome.
CONCLUSION
DBI acts as a strategic barrier against overactivation of phagophore maturation under nutrient-rich conditions, while triggering autophagy under nutrient-deficient conditions.
Topics: Humans; Carrier Proteins; Phosphatidylethanolamines; Autophagy; Nutrients; Serine
PubMed: 36280213
DOI: 10.1016/j.metabol.2022.155338