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1.
Nat Commun ; 14(1): 423, 2023 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-36702874

RESUMO

Auxin efflux through plasma-membrane-integral PIN-FORMED (PIN) carriers is essential for plant tissue organization and tightly regulated. For instance, a molecular rheostat critically controls PIN-mediated auxin transport in developing protophloem sieve elements of Arabidopsis roots. Plasma-membrane-association of the rheostat proteins, BREVIS RADIX (BRX) and PROTEIN KINASE ASSOCIATED WITH BRX (PAX), is reinforced by interaction with PHOSPHATIDYLINOSITOL-4-PHOSPHATE-5-KINASE (PIP5K). Genetic evidence suggests that BRX dampens autocrine signaling of CLAVATA3/EMBRYO SURROUNDING REGION-RELATED 45 (CLE45) peptide via its receptor BARELY ANY MERISTEM 3 (BAM3). How excess CLE45-BAM3 signaling interferes with protophloem development and whether it does so directly or indirectly remains unclear. Here we show that rheostat polarity is independent of PIN polarity, but interdependent with PIP5K. Catalytically inactive PIP5K confers rheostat polarity without reinforcing its localization, revealing a possible PIP5K scaffolding function. Moreover, PIP5K and PAX cooperatively control local PIN abundance. We further find that CLE45-BAM3 signaling branches via RLCK-VII/PBS1-LIKE (PBL) cytoplasmic kinases to destabilize rheostat localization. Our data thus reveal antagonism between CLE45-BAM3-PBL signaling and PIP5K that converges on auxin efflux regulation through dynamic control of PAX polarity. Because second-site bam3 mutation suppresses root as well as shoot phenotypes of pip5k mutants, CLE peptide signaling likely modulates phosphoinositide-dependent processes in various developmental contexts.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Fosfatidilinositóis/metabolismo , Raízes de Plantas/metabolismo , Arabidopsis/metabolismo , Ácidos Indolacéticos/metabolismo , Peptídeos/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Membrana/metabolismo
2.
Proc Natl Acad Sci U S A ; 120(5): e2212755120, 2023 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-36693100

RESUMO

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB), a disease that claims ~1.6 million lives annually. The current treatment regime is long and expensive, and missed doses contribute to drug resistance. Therefore, development of new anti-TB drugs remains one of the highest public health priorities. Mtb has evolved a complex cell envelope that represents a formidable barrier to antibiotics. The Mtb cell envelop consists of four distinct layers enriched for Mtb specific lipids and glycans. Although the outer membrane, comprised of mycolic acid esters, has been extensively studied, less is known about the plasma membrane, which also plays a critical role in impacting antibiotic efficacy. The Mtb plasma membrane has a unique lipid composition, with mannosylated phosphatidylinositol lipids (phosphatidyl-myoinositol mannosides, PIMs) comprising more than 50% of the lipids. However, the role of PIMs in the structure and function of the membrane remains elusive. Here, we used multiscale molecular dynamics (MD) simulations to understand the structure-function relationship of the PIM lipid family and decipher how they self-organize to shape the biophysical properties of mycobacterial plasma membranes. We assess both symmetric and asymmetric assemblies of the Mtb plasma membrane and compare this with residue distributions of Mtb integral membrane protein structures. To further validate the model, we tested known anti-TB drugs and demonstrated that our models agree with experimental results. Thus, our work sheds new light on the organization of the mycobacterial plasma membrane. This paves the way for future studies on antibiotic development and understanding Mtb membrane protein function.


Assuntos
Mycobacterium tuberculosis , Tuberculose , Humanos , Fosfatidilinositóis/metabolismo , Mycobacterium tuberculosis/metabolismo , Membrana Celular/metabolismo , Tuberculose/microbiologia , Antituberculosos/metabolismo
3.
Int J Mol Sci ; 24(2)2023 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-36674478

RESUMO

The Src homology 2 domain-containing inositol 5-phosphatase 1 (SHIP1) is known to dephosphorylate PtdIns(3,4,5)P3 into PtdIns(3,4)P2 and to interact with several signaling proteins though its docking functions. It has been shown to negatively regulate platelet adhesion and spreading on a fibrinogen surface and to positively regulate thrombus growth. In the present study, we have investigated its role during the early phase of platelet activation. Using confocal-based morphometric analysis, we found that SHIP1 is involved in the regulation of cytoskeletal organization and internal contractile activity in thrombin-activated platelets. The absence of SHIP1 has no significant impact on thrombin-induced Akt or Erk1/2 activation, but it selectively affects the RhoA/Rho-kinase pathway and myosin IIA relocalization to the cytoskeleton. SHIP1 interacts with the spectrin-based membrane skeleton, and its absence induces a loss of sustained association of integrins to this network together with a decrease in αIIbß3 integrin clustering following thrombin stimulation. This αIIbß3 integrin dynamics requires the contractile cytoskeleton under the control of SHIP1. RhoA activation, internal platelet contraction, and membrane skeleton integrin association were insensitive to the inhibition of PtdIns(3,4,5)P3 synthesis or SHIP1 phosphatase activity, indicating a role for the docking properties of SHIP1 in these processes. Altogether, our data reveal a lipid-independent function for SHIP1 in the regulation of the contractile cytoskeleton and integrin dynamics in platelets.


Assuntos
Integrina alfa2 , Integrina beta3 , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases , Ativação Plaquetária , Plaquetas/metabolismo , Integrina beta3/metabolismo , Fosfatidilinositóis/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Trombina/farmacologia , Trombina/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , Integrina alfa2/metabolismo
4.
Int J Mol Sci ; 24(2)2023 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-36674645

RESUMO

Arap3, a dual GTPase-activating protein (GAP) for the small GTPases Arf6 and RhoA, plays key roles in regulating a wide range of biological processes, including cancer cell invasion and metastasis. It is known that Arap3 is a PI3K effector that can bind directly to PI(3,4,5)P3, and the PI(3,4,5)P3-mediated plasma membrane recruitment is crucial for its function. However, the molecular mechanism of how the protein recognizes PI(3,4,5)P3 remains unclear. Here, using liposome pull-down and surface plasmon resonance (SPR) analysis, we found that the N-terminal first pleckstrin homology (PH) domain (Arap3-PH1) can interact with PI(3,4,5)P3 and, with lower affinity, with PI(4,5)P2. To understand how Arap3-PH1 and phosphoinositide (PIP) lipids interact, we solved the crystal structure of the Arap3-PH1 in the apo form and complex with diC4-PI(3,4,5)P3. We also characterized the interactions of Arap3-PH1 with diC4-PI(3,4,5)P3 and diC4-PI(4,5)P2 in solution by nuclear magnetic resonance (NMR) spectroscopy. Furthermore, we found overexpression of Arap3 could inhibit breast cancer cell invasion in vitro, and the PIPs-binding ability of the PH1 domain is essential for this function.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Ativadoras de GTPase , Fosfatidilinositóis , Humanos , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Ativadoras de GTPase/química , Invasividade Neoplásica , Fosfatos de Fosfatidilinositol/metabolismo , Ligação Proteica , Domínios Proteicos
5.
Nat Immunol ; 24(1): 136-147, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36581712

RESUMO

Hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) by phospholipase C-γ (PLCγ1) represents a critical step in T cell antigen receptor (TCR) signaling and subsequent thymocyte and T cell responses. PIP2 replenishment following its depletion in the plasma membrane (PM) is dependent on delivery of its precursor phosphatidylinositol (PI) from the endoplasmic reticulum (ER) to the PM. We show that a PI transfer protein (PITP), Nir3 (Pitpnm2), promotes PIP2 replenishment following TCR stimulation and is important for T cell development. In Nir3-/- T lineage cells, the PIP2 replenishment following TCR stimulation is slower. Nir3 deficiency attenuates calcium mobilization in double-positive (DP) thymocytes in response to weak TCR stimulation. This impaired TCR signaling leads to attenuated thymocyte development at TCRß selection and positive selection as well as diminished mature T cell fitness in Nir3-/- mice. This study highlights the importance of PIP2 replenishment mediated by PITPs at ER-PM junctions during TCR signaling.


Assuntos
Proteínas de Transferência de Fosfolipídeos , Transdução de Sinais , Camundongos , Animais , Proteínas de Transferência de Fosfolipídeos/metabolismo , Membrana Celular/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Fosfatidilinositóis/metabolismo
6.
Anal Biochem ; 663: 115019, 2023 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-36526022

RESUMO

Ras family GTPases (H/K/N-Ras) modulate numerous effectors, including the lipid kinase PI3K (phosphatidylinositol-3-kinase) that generates growth signal lipid PIP3 (phosphatidylinositol-3,4,5-triphosphate). Active GTP-Ras binds PI3K with high affinity, thereby stimulating PIP3 production. We hypothesize the affinity of this binding interaction could be significantly increased or decreased by Ras mutations at PI3K contact positions, with clinical implications since some Ras mutations at PI3K contact positions are disease-linked. To enable tests of this hypothesis, we have developed an approach combining UV spectral deconvolution, HPLC, and microscale thermophoresis to quantify the KD for binding. The approach measures the total Ras concentration, the fraction of Ras in the active state, and the affinity of active Ras binding to its docking site on PI3K Ras binding domain (RBD) in solution. The approach is illustrated by KD measurements for the binding of active H-Ras and representative mutants, each loaded with GTP or GMPPNP, to PI3Kγ RBD. The findings demonstrate that quantitation of the Ras activation state increases the precision of KD measurements, while also revealing that Ras mutations can increase (Q25L), decrease (D38E, Y40C), or have no effect (G13R) on PI3K binding affinity. Significant Ras affinity changes are predicted to alter PI3K regulation and PIP3 growth signals.


Assuntos
Fosfatidilinositol 3-Quinases , Proteínas ras , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/química , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas ras/química , Ligação Proteica , Guanosina Trifosfato/metabolismo , Fosfatidilinositóis
7.
Biochim Biophys Acta Biomembr ; 1864(1): 183780, 2022 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-34547252

RESUMO

The general segregation of a molecular class, lipids, from the pathways of cellular communication, via endo-membranes, has resulted in the over-simplification and misconceptions in deciphering cell signalling mechanisms. Mechanisms in signal transduction and protein activation require targeting of proteins to membranous compartments with a specific localised morphology and dynamics that are dependent on their lipid composition. Many posttranslational events define cellular behaviours and without the active role of membranous compartments these events lead to various dysregulations of the signalling pathways. We summarise the key findings, using tools such as the rapalogue dimerisation, in the structural roles and signalling of the inter-related phosphoinositide lipids and their derivative, diacylglycerol, in the regulation of nuclear envelope biogenesis and other subcellular compartments such as the nucleoplasmic reticulum.


Assuntos
Comunicação Celular/genética , Lipídeos de Membrana/genética , Fosfatidilinositóis/genética , Transdução de Sinais/genética , Membrana Nuclear/genética
8.
Zhonghua Wei Zhong Bing Ji Jiu Yi Xue ; 34(11): 1161-1166, 2022 Nov.
Artigo em Chinês | MEDLINE | ID: mdl-36567559

RESUMO

OBJECTIVE: To investigate the effect of digoxin on bleomycin-induced pulmonary fibrosis in mice, and investigate its possible mechanism through in vitro and in vivo experiments. METHODS: (1) In vivo experiment: 60 C57/BL6J mice were randomly divided into control group, pulmonary fibrosis model group (model group), pirfenidone (300 mg/kg) group, digoxin 1.0 mg/kg and 0.2 mg/kg groups, with 12 mice in each group. The pulmonary fibrosis model of mice was reproduced by single intratracheal infusion of bleomycin (5 mg/kg). The control group was given the same amount of sterile normal saline. From the next day after modeling, each group was received corresponding drugs by intragastric administration once a day for 28 days. Control group and model group were given the same amount of normal saline. The mice were sacrificed and the lung tissue was collected to detect the lung coefficient. After hematoxylin-eosin (HE) and Masson staining, the lung tissue morphology and collagen changes were observed under light microscope. Immunohistochemistry was used to detect the positive expressions of α-smooth muscle actin (α-SMA) and extracellular matrix (ECM) collagen (COL-I and COL-III) in lung tissue. The protein expressions of ECM fibronectin (FN), transforming growth factor-ß (TGF-ß) and phosphorylation of Smad3 (p-Smad3) in lung tissue were detected by Western blotting. (2) In vitro experiment: human embryonic lung fibroblast-1 (HFL-1) cells were cultured and divided into blank control group, fibroblast activation model group (model group), pirfenidone (2.5 mmol/L) group and digoxin 100 nmol/L and 50 nmol/L groups when cell density reached 70%-90%. After 3-hour treatment with corresponding drugs, except blank control group, the other groups were treated with TGF-ß for 48 hours to establish fibroblast activation model. The expressions of α-SMA, FN and p-Smad3 proteins and the phosphorylations of phosphatidylinositol-3-kinase (PI3K)/Akt pathway proteins PI3K and Akt (p-PI3K, p-Akt) were detected by Western blotting. RESULTS: (1) In vivo, compared with the control group, the alveolar structure of mice in the model group was significantly damaged, a large number of inflammatory cells infiltrated, collagen deposition in the lung interstitium was increased, the deposition of ECM in the lung tissue was also increased, and the expressions of α-SMA, FN, TGF-ß and p-Smad3 protein were increased, indicating that the model of bleomycin-induced pulmonary fibrosis in mice was successfully prepared. Compared with the model group, digoxin significantly inhibited airway inflammation and collagen fiber deposition, reduced ECM deposition, and decreased the protein expressions of α-SMA, FN, TGF-ß and p-Smad3, while the effect was better than that of the pirfenidone group, and the digoxin 1.0 mg/kg group had a better effect except FN [α-SMA (A value): 5.37±1.10 vs. 9.51±1.66, TGF-ß protein (TGF-ß/GAPDH): 0.09±0.04 vs. 0.33±0.23, p-Smad3 protein (p-Smad3/GAPDH): 0.05±0.01 vs. 0.20±0.07, all P < 0.01]. (2) In vitro, compared with the blank control group, the expressions of FN, α-SMA, p-Smad3 and PI3K/Akt signaling proteins in the model group were increased, indicating that the fibroblast activation model induced by TGF-ß was successfully reproduced. Compared with the model group, digoxin significantly inhibited fibroblast activation, and decreased the expressions of FN, α-SMA, p-Smad3, and PI3K/Akt pathway proteins, moreover, the effect was better than that of the pirfenidone group, and decreased FN, SMA and p-Akt protein expressions were more obvious in digoxin 100 nmol/L group [FN protein (FN/GAPDH): 0.21±0.15 vs. 0.88±0.22, α-SMA protein (α-SMA/GAPDH): 0.20±0.01 vs. 0.50±0.08, p-Akt protein (p-Akt/GAPDH): 0.30±0.01 vs. 0.65±0.10, all P < 0.01]. CONCLUSIONS: Digoxin could suppress the pulmonary fibrosis in mice induced by bleomycin, which might be associated with the regulation of fibroblast activation via suppressing PI3K/Akt signaling pathway in a dose-dependent manner.


Assuntos
Fibrose Pulmonar , Camundongos , Humanos , Animais , Fibrose Pulmonar/tratamento farmacológico , Fibrose Pulmonar/induzido quimicamente , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína Smad3/metabolismo , Proteína Smad3/farmacologia , Digoxina/metabolismo , Digoxina/farmacologia , Digoxina/uso terapêutico , Fosfatidilinositol 3-Quinases/metabolismo , Solução Salina/uso terapêutico , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Fator de Crescimento Transformador beta/uso terapêutico , Fibroblastos/metabolismo , Fibroblastos/patologia , Transdução de Sinais , Bleomicina/metabolismo , Bleomicina/farmacologia , Bleomicina/uso terapêutico , Colágeno/metabolismo , Colágeno/farmacologia , Colágeno/uso terapêutico , Fosfatidilinositóis/metabolismo , Fosfatidilinositóis/farmacologia , Fosfatidilinositóis/uso terapêutico , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta1/farmacologia
9.
Science ; 378(6625): eabq5209, 2022 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-36520888

RESUMO

Cells respond to fluctuating nutrient supply by adaptive changes in organelle dynamics and in metabolism. How such changes are orchestrated on a cell-wide scale is unknown. We show that endosomal signaling lipid turnover by MTM1, a phosphatidylinositol 3-phosphate [PI(3)P] 3-phosphatase mutated in X-linked centronuclear myopathy in humans, controls mitochondrial morphology and function by reshaping the endoplasmic reticulum (ER). Starvation-induced endosomal recruitment of MTM1 impairs PI(3)P-dependent contact formation between tubular ER membranes and early endosomes, resulting in the conversion of ER tubules into sheets, the inhibition of mitochondrial fission, and sustained oxidative metabolism. Our results unravel an important role for early endosomal lipid signaling in controlling ER shape and, thereby, mitochondrial form and function to enable cells to adapt to fluctuating nutrient environments.


Assuntos
Retículo Endoplasmático , Endossomos , Mitocôndrias , Dinâmica Mitocondrial , Fosfatos de Fosfatidilinositol , Fosfatidilinositóis , Proteínas Tirosina Fosfatases não Receptoras , Humanos , Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Mitocôndrias/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilinositóis/metabolismo , Transdução de Sinais , Proteínas Tirosina Fosfatases não Receptoras/genética , Proteínas Tirosina Fosfatases não Receptoras/metabolismo
10.
Cells ; 11(24)2022 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-36552741

RESUMO

The plasma membrane of eukaryotic cells is composed of a large number of lipid species that are laterally segregated into functional domains as well as asymmetrically distributed between the outer and inner leaflets. Additionally, the spatial distribution and organization of these lipids dramatically change in response to various cellular states, such as cell division, differentiation, and apoptosis. Division of one cell into two daughter cells is one of the most fundamental requirements for the sustenance of growth in all living organisms. The successful completion of cytokinesis, the final stage of cell division, is critically dependent on the spatial distribution and organization of specific lipids. In this review, we discuss the properties of various lipid species associated with cytokinesis and the mechanisms involved in their polarization, including forward trafficking, endocytic recycling, local synthesis, and cortical flow models. The differences in lipid species requirements and distribution in mitotic vs. male meiotic cells will be discussed. We will concentrate on sphingolipids and phosphatidylinositols because their transbilayer organization and movement may be linked via the cytoskeleton and thus critically regulate various steps of cytokinesis.


Assuntos
Citocinese , Fosfatidilinositóis , Masculino , Humanos , Citocinese/fisiologia , Divisão Celular , Membrana Celular/metabolismo , Transporte Biológico , Fosfatidilinositóis/metabolismo
11.
Front Cell Infect Microbiol ; 12: 1012533, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36389142

RESUMO

Shigella, the causative agent of bacillary dysentery, subvert cytoskeletal and trafficking processes to invade and replicate in epithelial cells using an arsenal of bacterial effectors translocated through a type III secretion system. Here, we review the various roles of the type III effector IpgD, initially characterized as phosphatidylinositol 4,5 bisphosphate (PI4,5P2) 4-phosphatase. By decreasing PI4,5P2 levels, IpgD triggers the disassembly of cortical actin filaments required for bacterial invasion and cell migration. PI5P produced by IpgD further stimulates signaling pathways regulating cell survival, macropinosome formation, endosomal trafficking and dampening of immune responses. Recently, IpgD was also found to exhibit phosphotransferase activity leading to PI3,4P2 synthesis adding a new flavor to this multipotent bacterial enzyme. The substrate of IpgD, PI4,5P2 is also the main substrate hydrolyzed by endogenous phospholipases C to produce inositoltriphosphate (InsP3), a major Ca2+ second messenger. Hence, beyond the repertoire of effects associated with the direct diversion of phoshoinositides, IpgD indirectly down-regulates InsP3-mediated Ca2+ release by limiting InsP3 production. Furthermore, IpgD controls the intracellular lifestyle of Shigella promoting Rab8/11 -dependent recruitment of the exocyst at macropinosomes to remove damaged vacuolar membrane remnants and promote bacterial cytosolic escape. IpgD thus emerges as a key bacterial effector for the remodeling of host cell membranes.


Assuntos
Disenteria Bacilar , Shigella , Humanos , Fosfatidilinositóis/metabolismo , Shigella flexneri/metabolismo , Disenteria Bacilar/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo
12.
Elife ; 112022 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-36374183

RESUMO

High-voltage-activated Ca2+ (CaV) channels that adjust Ca2+ influx upon membrane depolarization are differentially regulated by phosphatidylinositol 4,5-bisphosphate (PIP2) in an auxiliary CaV ß subunit-dependent manner. However, the molecular mechanism by which the ß subunits control the PIP2 sensitivity of CaV channels remains unclear. By engineering various α1B and ß constructs in tsA-201 cells, we reported that at least two PIP2-binding sites, including the polybasic residues at the C-terminal end of I-II loop and the binding pocket in S4II domain, exist in the CaV2.2 channels. Moreover, they were distinctly engaged in the regulation of channel gating depending on the coupled CaV ß2 subunits. The membrane-anchored ß subunit abolished the PIP2 interaction of the phospholipid-binding site in the I-II loop, leading to lower PIP2 sensitivity of CaV2.2 channels. By contrast, PIP2 interacted with the basic residues in the S4II domain of CaV2.2 channels regardless of ß2 isotype. Our data demonstrated that the anchoring properties of CaV ß2 subunits to the plasma membrane determine the biophysical states of CaV2.2 channels by regulating PIP2 coupling to the nonspecific phospholipid-binding site in the I-II loop.


Assuntos
Canais de Cálcio Tipo N , Fosfatidilinositóis , Canais de Cálcio Tipo N/genética , Canais de Cálcio Tipo N/metabolismo , Membrana Celular/metabolismo , Fosfatidilinositóis/metabolismo , Sítios de Ligação
13.
Cell ; 185(24): 4560-4573.e19, 2022 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-36368322

RESUMO

Binding of arrestin to phosphorylated G protein-coupled receptors (GPCRs) is crucial for modulating signaling. Once internalized, some GPCRs remain complexed with ß-arrestins, while others interact only transiently; this difference affects GPCR signaling and recycling. Cell-based and in vitro biophysical assays reveal the role of membrane phosphoinositides (PIPs) in ß-arrestin recruitment and GPCR-ß-arrestin complex dynamics. We find that GPCRs broadly stratify into two groups, one that requires PIP binding for ß-arrestin recruitment and one that does not. Plasma membrane PIPs potentiate an active conformation of ß-arrestin and stabilize GPCR-ß-arrestin complexes by promoting a fully engaged state of the complex. As allosteric modulators of GPCR-ß-arrestin complex dynamics, membrane PIPs allow for additional conformational diversity beyond that imposed by GPCR phosphorylation alone. For GPCRs that require membrane PIP binding for ß-arrestin recruitment, this provides a mechanism for ß-arrestin release upon translocation of the GPCR to endosomes, allowing for its rapid recycling.


Assuntos
Arrestinas , Fosfatidilinositóis , beta-Arrestinas/metabolismo , Fosfatidilinositóis/metabolismo , Arrestinas/metabolismo , beta-Arrestina 1/metabolismo , Receptores Acoplados a Proteínas G/metabolismo
14.
Commun Biol ; 5(1): 1259, 2022 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-36396757

RESUMO

The plasmalemmal norepinephrine transporter (NET) regulates cardiovascular sympathetic activity by clearing extracellular norepinephrine in the synaptic cleft. Here, we investigate the subunit stoichiometry and function of NET using single-molecule fluorescence microscopy and flux assays. In particular, we show the effect of phosphatidylinositol 4,5-bisphosphate (PIP2) on NET oligomerization and efflux. NET forms monomers (~60%) and dimers (~40%) at the plasma membrane. PIP2 depletion results in a decrease in the average oligomeric state and decreases NET-mediated substrate efflux while not affecting substrate uptake. Mutation of the putative PIP2 binding residues R121, K334, and R440 to alanines does not affect NET dimerization but results in decreased substrate efflux that is not altered upon PIP2 depletion; this indicates that PIP2 interactions with these residues affect NET-mediated efflux. A dysregulation of norepinephrine and PIP2 signaling have both been implicated in neuropsychiatric and cardiovascular diseases. This study provides evidence that PIP2 directly regulates NET organization and function.


Assuntos
Proteínas da Membrana Plasmática de Transporte de Norepinefrina , Fosfatidilinositóis , Proteínas da Membrana Plasmática de Transporte de Norepinefrina/genética , Dimerização , Transporte Biológico , Fosfatos de Inositol , Norepinefrina
15.
Medicine (Baltimore) ; 101(45): e31501, 2022 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-36397361

RESUMO

Previously, a case series study was conducted on our part in which 5 patients with Graves' disease (GD) were collected from a 3-generation family to screen for susceptibility genes responsible for GD. The single nucleotide variants of Microtubule-associated protein 7 domain containing 2 c. 452C > T, p. Ala151Val, Solute carrier family 1 member 7 c. 1204C > T, p. Arg402Cys, tumor necrosis factor receptor-associated factor 3 interacting protein 3 (TRAF3IP3) c. 209A > T, p. Asn70Ile, protein tyrosine phosphatase receptor type B (PTPRB) c. 3472A > G, p. Ser1158Gly, Phosphoinositide-3-kinase regulatory subunit 3 c. 121C > T, p. Pro41Ser, disrupted in schizophrenia 1 (DISC1), c. 1591G > C p. Gly531Arg were associated with the familial GD. We then further confirmed these variants and investigated whether other mutations render susceptibility to GD. The case-control study collected patients with sporadic GD or no GD family history. A snapshot program was used for genotyping the selected SNPs in 235 GD patients (GD group 1) and 284 healthy patients (control group). Furthermore, another 184 GD patients were recruited (GD group 2) to sequence the specified exons of these genes. The sequenced data was compared with Chinese Millionome Database (CMDB). Several variants of PTPRB, phosphoinositide-3-kinase regulatory subunit 3, TRAF3IP3, and DISC1 were found in GD group 2 but not in CMDB. Moreover, the allele frequency of SNP rs2076150 (TRAF3IP3) and rs2492367 DISC1 in GD group 2 was significantly higher than that of in CMDB (all P < .05). When the control group or CMDB was set as a reference group, a significantly higher frequency in alter allele C of SNP rs186466118 PTPRB was observed in GD group 1 and GD group (constituted by GD group 1 and GD group 2). Equally importantly, there was a correlation between the allele C of SNP rs186466118 and the increased risk of GD susceptibility (all P < .05). PTPRB, TRAF3IP3, and DISC1 may be susceptibility genes for GD, and more variants of PTPRB, TRAF3IP3, and DISC1 were found in GD patients.


Assuntos
Predisposição Genética para Doença , Doença de Graves , Humanos , Estudos de Casos e Controles , China , Doença de Graves/genética , Proteínas do Tecido Nervoso/genética , Fosfatidilinositóis , Monoéster Fosfórico Hidrolases , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/genética
16.
Front Immunol ; 13: 982383, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36341455

RESUMO

Naive B cells use the chemokine receptor CXCR5 to enter B cell follicles, where they scan CXCL13-expressing ICAM-1+ VCAM-1+ follicular dendritic cells (FDCs) for the presence of antigen. CXCL13-CXCR5-mediated motility is mainly driven by the Rac guanine exchange factor DOCK2, which contains a binding domain for phosphoinositide-3,4,5-triphosphate (PIP3) and other phospholipids. While p110δ, the catalytic subunit of the class IA phosphoinositide-3-kinase (PI3K) δ, contributes to CXCR5-mediated B cell migration, the precise interdependency of DOCK2, p110δ, or other PI3K family members during this process remains incompletely understood. Here, we combined in vitro chemotaxis assays and in vivo imaging to examine the contribution of these two factors during murine naïve B cell migration to CXCL13. Our data confirm that p110δ is the main catalytic subunit mediating PI3K-dependent migration downstream CXCR5, whereas it does not contribute to chemotaxis triggered by CXCR4 or CCR7, two other chemokine receptors expressed on naïve B cells. The contribution of p110δ activity to CXCR5-driven migration was complementary to that of DOCK2, and pharmacological or genetic interference with both pathways completely abrogated B cell chemotaxis to CXCL13. Intravital microscopy of control and gene-deficient B cells migrating on FDCs confirmed that lack of DOCK2 caused a profound migration defect, whereas p110δ contributed to cell speed and directionality. B cells lacking active p110δ also displayed defective adhesion to ICAM-1; yet, their migration impairment was maintained on ICAM-1-deficient FDCs. In sum, our data uncover two complementary signaling pathways mediated by DOCK2 and p110δ, which enable CXCR5-driven naïve B cell examination of FDCs.


Assuntos
Molécula 1 de Adesão Intercelular , Fosfatidilinositol 3-Quinases , Camundongos , Animais , Molécula 1 de Adesão Intercelular/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Receptores CXCR5/metabolismo , Transdução de Sinais , Fatores de Troca do Nucleotídeo Guanina/genética , Quimiotaxia de Leucócito , Receptores de Quimiocinas , Fosfatidilinositóis , Proteínas Ativadoras de GTPase
17.
Biochem J ; 479(21): 2311-2325, 2022 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-36367756

RESUMO

In the almost 70 years since the first hints of its existence, the phosphoinositide, phosphatidyl-D-myo-inositol 4,5-bisphosphate has been found to be central in the biological regulation of plasma membrane (PM) function. Here, we provide an overview of the signaling, transport and structural roles the lipid plays at the cell surface in animal cells. These include being substrate for second messenger generation, direct modulation of receptors, control of membrane traffic, regulation of ion channels and transporters, and modulation of the cytoskeleton and cell polarity. We conclude by re-evaluating PI(4,5)P2's designation as a signaling molecule, instead proposing a cofactor role, enabling PM-selective function for many proteins.


Assuntos
Fosfatidilinositóis , Transdução de Sinais , Animais , Membrana Celular/metabolismo , Fosfatidilinositóis/metabolismo , Canais Iônicos/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo
18.
PLoS Pathog ; 18(11): e1010922, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36318587

RESUMO

Phosphoinositides are important second messengers that regulate key cellular processes in eukaryotes. While it is known that a single phosphoinositol-3 kinase (PI3K) catalyses the formation of 3'-phosphorylated phosphoinositides (PIPs) in apicomplexan parasites like Plasmodium and Toxoplasma, how its activity and PI3P formation is regulated has remained unknown. Present studies involving a unique Vps15 like protein (TgVPS15) in Toxoplasma gondii provides insight into the regulation of phosphatidyl-3-phosphate (PI3P) generation and unravels a novel pathway that regulates parasite development. Detailed investigations suggested that TgVPS15 regulates PI3P formation in Toxoplasma gondii, which is important for the inheritance of the apicoplast-a plastid like organelle present in most apicomplexans and parasite replication. Interestingly, TgVPS15 also regulates autophagy in T. gondii under nutrient-limiting conditions as it promotes autophagosome formation. For both these processes, TgVPS15 uses PI3P-binding protein TgATG18 and regulates trafficking and conjugation of TgATG8 to the apicoplast and autophagosomes, which is important for biogenesis of these organelles. TgVPS15 has a protein kinase domain but lacks several key residues conserved in conventional protein kinases. Interestingly, two critical residues in its active site are important for PI3P formation and parasitic functions of this kinase. Collectively, these studies unravel a signalling cascade involving TgVPS15, a novel effector of PI3-kinase in T. gondii and possibly other Apicomplexa, that regulate critical processes like apicoplast biogenesis and autophagy.


Assuntos
Apicoplastos , Parasitos , Toxoplasma , Animais , Apicoplastos/fisiologia , Toxoplasma/metabolismo , Autofagia , Autofagossomos/metabolismo , Parasitos/metabolismo , Fosfatidilinositóis/metabolismo , Proteínas de Protozoários/metabolismo
19.
Viruses ; 14(11)2022 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-36423118

RESUMO

The fully assembled influenza A virus (IAV) has on its surface the highest density of a single membrane protein found in nature-the glycoprotein hemagglutinin (HA) that mediates viral binding, entry, and assembly. HA clusters at the plasma membrane of infected cells, and the HA density (number of molecules per unit area) of these clusters correlates with the infectivity of the virus. Dense HA clusters are considered to mark the assembly site and ultimately lead to the budding of infectious IAV. The mechanism of spontaneous HA clustering, which occurs with or without other viral components, has not been elucidated. Using super-resolution fluorescence photoactivation localization microscopy (FPALM), we have previously shown that these HA clusters are interdependent on phosphatidylinositol 4,5-biphosphate (PIP2). Here, we show that the IAV matrix protein M1 co-clusters with PIP2, visualized using the pleckstrin homology domain. We find that cetylpyridinium chloride (CPC), which is a positively charged quaternary ammonium compound known for its antibacterial and antiviral properties at millimolar concentrations, disrupts M1 clustering and M1-PIP2 co-clustering at micromolar concentrations well below the critical micelle concentration (CMC). CPC also disrupts the co-clustering of M1 with HA at the plasma membrane, suggesting the role of host cell PIP2 clusters as scaffolds for gathering and concentrating M1 and HA to achieve their unusually high cluster densities in the IAV envelope.


Assuntos
Vírus da Influenza A , Influenza Humana , Humanos , Hemaglutininas/metabolismo , Fosfatidilinositóis/metabolismo , Influenza Humana/metabolismo , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Montagem de Vírus , Membrana Celular/metabolismo , Vírus da Influenza A/fisiologia
20.
Nutrients ; 14(20)2022 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-36297104

RESUMO

As centenarians provide a paradigm of healthy aging, investigating the comprehensive metabolic profiles of healthy centenarians is of utmost importance for the pursuit of health and longevity. However, relevant reports, especially studies considering the dietary influence on metabolism, are still limited, mostly lacking the guidance of a model of healthy aging. Therefore, exploring the signatures of the integrative metabolic profiles of the healthy centenarians from a famous longevous region, Bama County, China, should be an effective way. The global metabolome in urine and the short-chain fatty acids (SCFAs) in the feces of 30 healthy centenarians and 31 elderly people aged 60-70 from the longevous region were analyzed by non-targeted metabolomics combined with metabolic target analysis. The results showed that the characteristic metabolites related to longevity were mostly summarized into phosphatidylserine, lyso-phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol, bile acids, and amino acids (p < 0.05). Six metabolic pathways were found significant relevant to longevity. Furthermore, acetic acid, propionic acid, butyric acid, valeric acid, and total SCFA were significantly increased in the centenarian group (p < 0.05) and were also positively associated with the dietary fiber intake (p < 0.01). It was age-accompanied and diet-associated remodeling of phospholipid, amino acid, and SCFA metabolism that expressed the unique metabolic signatures related to exceptional longevity. This metabolic remodeling is suggestive of cognitive benefits, better antioxidant capacity, the attenuation of local inflammation, and health-span-promoting processes, which play a critical and positive role in shaping healthy aging.


Assuntos
Longevidade , Propionatos , Idoso de 80 Anos ou mais , Idoso , Humanos , Aminoácidos , Fosfatidiletanolaminas , Fosfolipídeos , Centenários , Fosfatidilserinas , Antioxidantes , Dieta , China , Ácidos Graxos Voláteis , Ácido Butírico , Fibras na Dieta , Acetatos , Fosfatidilinositóis , Ácidos e Sais Biliares , Fosfatidilcolinas
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