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1.
Plant Sci ; 313: 111087, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34763872

RESUMO

Four members of phosphoinositide-specific phospholipase C (PI-PLC) are predicted in rice genome. Although the involvement of OsPLC1 and OsPLC4 in the responses of rice to salt and drought stresses has been documented, the role of OsPLC3 in which, yet, is elusive. Here, we report that OsPLC3 was ubiquitously expressed in various tissues during the development of rice. The expression of YFP-tagged OsPLC3 was observed at the plasma membrane (PM), cytoplasm and nucleus of rice protoplasts, onion epidermal cells and tobacco leaves. The catalytic activity of OsPLC3 was measured using the thin-layer chromatography (TLC) method. The inhibition of OsPLC3 expression was detected in the treatments of NaCl and mannitol. Overexpression (OE) of OsPLC3 produced plants showing more sensitive to osmotic stresses when they were compared to the wild-type (HJ) and osplc3 mutants, the phenomena such as decreased plant fresh weight and increased water loss rate (WLR) were observed. Under the treatment of NaCl or mannitol, expressions of a subset osmotic stress-related genes were altered, in both OE and osplc3 mutant lines. In addition, the expressions and the enzyme activities of reactive oxygen species (ROS) scavengers were significantly decreased in OE lines, leading to over-accumulation of ROS together with less osmotic adjustment substances including proline, soluble sugars and soluble proteins in OE plants which caused the growth inhibition. Thus, our results suggested that, via modulating ROS homeostasis, OsPLC3 is involved in responses to the osmotic stress in rice.


Assuntos
Homeostase/fisiologia , Oryza/crescimento & desenvolvimento , Oryza/genética , Oryza/metabolismo , Pressão Osmótica/fisiologia , Fosfatidilinositóis/metabolismo , Fosfolipases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Produtos Agrícolas/genética , Produtos Agrícolas/crescimento & desenvolvimento , Produtos Agrícolas/metabolismo , Fosfatidilinositóis/genética , Fosfolipases/genética
2.
Nat Commun ; 12(1): 6065, 2021 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-34663803

RESUMO

Different types of cellular membranes have unique lipid compositions that are important for their functional identity. PI(4,5)P2 is enriched in the plasma membrane where it contributes to local activation of key cellular events, including actomyosin contraction and cytokinesis. However, how cells prevent PI(4,5)P2 from accumulating in intracellular membrane compartments, despite constant intermixing and exchange of lipid membranes, is poorly understood. Using the C. elegans early embryo as our model system, we show that the evolutionarily conserved lipid transfer proteins, PDZD-8 and TEX-2, act together with the PI(4,5)P2 phosphatases, OCRL-1 and UNC-26/synaptojanin, to prevent the build-up of PI(4,5)P2 on endosomal membranes. In the absence of these four proteins, large amounts of PI(4,5)P2 accumulate on endosomes, leading to embryonic lethality due to ectopic recruitment of proteins involved in actomyosin contractility. PDZD-8 localizes to the endoplasmic reticulum and regulates endosomal PI(4,5)P2 levels via its lipid harboring SMP domain. Accumulation of PI(4,5)P2 on endosomes is accompanied by impairment of their degradative capacity. Thus, cells use multiple redundant systems to maintain endosomal PI(4,5)P2 homeostasis.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Endocitose/fisiologia , Endossomos/metabolismo , Proteínas de Membrana/metabolismo , Actomiosina/metabolismo , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Membrana Celular/metabolismo , Citocinese , Desenvolvimento Embrionário , Retículo Endoplasmático/metabolismo , Homeostase , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso , Fosfatidilinositóis , Monoéster Fosfórico Hidrolases
3.
Dev Cell ; 56(18): 2542-2544, 2021 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-34582770

RESUMO

Although contractile processes, from tissue invagination to cell intercalation, utilize diverse ratcheting mechanisms, little is known about how ratcheting becomes engaged at specific cell surfaces. In this issue of Developmental Cell, Maio et al. demonstrate that PI(3,4,5)P3 is a paramount regulator of the Sbf/RabGEF-Rab35 ratchet mechanism.


Assuntos
Fosfatidilinositóis , Membrana Celular , Constrição
4.
Dev Cell ; 56(18): 2579-2591.e4, 2021 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-34525342

RESUMO

Force generation in epithelial tissues is often pulsatile, with actomyosin networks generating contractile forces before cyclically disassembling. This pulsed nature of cytoskeletal forces implies that there must be ratcheting mechanisms that drive processive transformations in cell shape. Previous work has shown that force generation is coordinated with endocytic remodeling; however, how ratcheting becomes engaged at specific cell surfaces remains unclear. Here, we report that PtdIns(3,4,5)P3 is a critical lipid-based cue for ratcheting engagement. The Sbf RabGEF binds to PIP3, and disruption of PIP3 reveals a dramatic switching behavior in which medial ratcheting is activated and epithelial cells begin globally constricting apical surfaces. PIP3 enrichments are developmentally regulated, with mesodermal cells having high apical PIP3 while germband cells have higher interfacial PIP3. Finally, we show that JAK/STAT signaling constitutes a second pathway that combinatorially regulates Sbf/Rab35 recruitment. Our results elucidate a complex lipid-dependent regulatory machinery that directs ratcheting engagement in epithelial tissues.


Assuntos
Actomiosina/metabolismo , Forma Celular/fisiologia , Células Epiteliais/metabolismo , Morfogênese/fisiologia , Fosfatidilinositóis/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Membrana Celular/metabolismo , Polaridade Celular/fisiologia , Citoesqueleto/metabolismo , Drosophila , Proteínas de Drosophila/metabolismo , Epitélio/metabolismo
5.
BMC Plant Biol ; 21(1): 365, 2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34380415

RESUMO

BACKGROUND: Kiwifruit (Actinidia Lindl.) is considered an important fruit species worldwide. Due to its temperate origin, this species is highly vulnerable to freezing injury while under low-temperature stress. To obtain further knowledge of the mechanism underlying freezing tolerance, we carried out a hybrid transcriptome analysis of two A. arguta (Actinidi arguta) genotypes, KL and RB, whose freezing tolerance is high and low, respectively. Both genotypes were subjected to - 25 °C for 0 h, 1 h, and 4 h. RESULTS: SMRT (single-molecule real-time) RNA-seq data were assembled using the de novo method, producing 24,306 unigenes with an N50 value of 1834 bp. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of DEGs showed that they were involved in the 'starch and sucrose metabolism', the 'mitogen-activated protein kinase (MAPK) signaling pathway', the 'phosphatidylinositol signaling system', the 'inositol phosphate metabolism', and the 'plant hormone signal transduction'. In particular, for 'starch and sucrose metabolism', we identified 3 key genes involved in cellulose degradation, trehalose synthesis, and starch degradation processes. Moreover, the activities of beta-GC (beta-glucosidase), TPS (trehalose-6-phosphate synthase), and BAM (beta-amylase), encoded by the abovementioned 3 key genes, were enhanced by cold stress. Three transcription factors (TFs) belonging to the AP2/ERF, bHLH (basic helix-loop-helix), and MYB families were involved in the low-temperature response. Furthermore, weighted gene coexpression network analysis (WGCNA) indicated that beta-GC, TPS5, and BAM3.1 were the key genes involved in the cold response and were highly coexpressed together with the CBF3, MYC2, and MYB44 genes. CONCLUSIONS: Cold stress led various changes in kiwifruit, the 'phosphatidylinositol signaling system', 'inositol phosphate metabolism', 'MAPK signaling pathway', 'plant hormone signal transduction', and 'starch and sucrose metabolism' processes were significantly affected by low temperature. Moreover, starch and sucrose metabolism may be the key pathway for tolerant kiwifruit to resist low temperature damages. These results increase our understanding of the complex mechanisms involved in the freezing tolerance of kiwifruit under cold stress and reveal a series of candidate genes for use in breeding new cultivars with enhanced freezing tolerance.


Assuntos
Aclimatação/genética , Actinidia/genética , Actinidia/fisiologia , Congelamento , Regulação da Expressão Gênica de Plantas , Frutas/genética , Frutas/fisiologia , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Sistema de Sinalização das MAP Quinases , Anotação de Sequência Molecular , Fosfatidilinositóis/metabolismo , Melhoramento Vegetal , Reguladores de Crescimento de Plantas/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Amido/metabolismo , Sacarose/metabolismo
6.
Int J Mol Sci ; 22(15)2021 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-34361045

RESUMO

Phosphoinositides (PIs) play important roles in the structure and function of the brain. Associations between PIs and the pathophysiology of schizophrenia have been studied. However, the significance of the PI metabolic pathway in the pathology of schizophrenia is unknown. We examined the expression of PI signaling-associated proteins in the postmortem brain of schizophrenia patients. Protein expression levels of phosphatidylinositol 4-phosphate 5-kinase type-1 gamma (PIP5K1C), phosphatidylinositol 4-kinase alpha (PIK4CA, also known as PIK4A), phosphatase and tensin homolog deleted from chromosome 10 (PTEN), protein kinase B (Akt), and glycogen synthase kinase 3ß (GSK3ß) were measured using enzyme-linked immunosorbent assays and multiplex fluorescent bead-based immunoassays of the prefrontal cortex (PFC) of postmortem samples from 23 schizophrenia patients and 47 normal controls. We also examined the association between PIK4CA expression and its genetic variants in the same brain samples. PIK4CA expression was lower, whereas Akt expression was higher, in the PFC of schizophrenia patients than in that of controls; PIP5K1C, PTEN, and GSK3ß expression was not different. No single-nucleotide polymorphism significantly affected protein expression. We identified molecules involved in the pathology of schizophrenia via this lipid metabolic pathway. These results suggest that PIK4CA is involved in the mechanism underlying the pathogenesis of schizophrenia and is a potential novel therapeutic target.


Assuntos
Fosfatidilinositóis/metabolismo , Córtex Pré-Frontal/metabolismo , Esquizofrenia/metabolismo , 1-Fosfatidilinositol 4-Quinase/metabolismo , Idoso , Feminino , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais
7.
J Phys Chem B ; 125(33): 9547-9556, 2021 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-34396776

RESUMO

The binding of phosphatidylinositol 4,5-bisphosphate (PIP2) to the ion channel transient receptor potential vanilloid 5 (TRPV5) is critical for its function. We use atomically detailed simulations and the milestoning theory to compute the free energy profile and the kinetics of PIP2 binding to TRPV5. We estimate the rate of binding and the impact of the protonation state on the process. Several channel residues are identified as influential in the association event and will be interesting targets for mutation analysis. Our simulations reveal that PIP2 binds to TRPV5 in an unprotonated state and is protonated in the membrane. The switch between the protonation state of PIP2 is modeled as a diabatic transition and occurs about halfway through the reaction.


Assuntos
Fosfatidilinositol 4,5-Difosfato , Fosfatidilinositóis , Fenômenos Biofísicos , Cinética , Termodinâmica
8.
Nat Commun ; 12(1): 4838, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34376698

RESUMO

Macropinosomes are formed by shaping actin-rich plasma membrane ruffles into large intracellular organelles in a phosphatidylinositol 3-kinase (PI3K)-coordinated manner. Here, we utilize lattice lightsheet microscopy and image visualization methods to map the three-dimensional structure and dynamics of macropinosome formation relative to PI3K activity. We show that multiple ruffling morphologies produce macropinosomes and that the majority form through collisions of adjacent PI3K-rich ruffles. By combining multiple volumetric representations of the plasma membrane structure and PI3K products, we show that PI3K activity begins early throughout the entire ruffle volume and continues to increase until peak activity concentrates at the base of the ruffle after the macropinosome closes. Additionally, areas of the plasma membrane rich in ruffling had increased PI3K activity and produced many macropinosomes of various sizes. Pharmacologic inhibition of PI3K activity had little effect on the rate and morphology of membrane ruffling, demonstrating that early production of 3'-phosphoinositides within ruffles plays a minor role in regulating their morphology. However, 3'-phosphoinositides are critical for the fusogenic activity that seals ruffles into macropinosomes. Taken together, these data indicate that local PI3K activity is amplified in ruffles and serves as a priming mechanism for closure and sealing of ruffles into macropinosomes.


Assuntos
Membrana Celular/metabolismo , Microscopia de Fluorescência/métodos , Fosfatidilinositol 3-Quinases/metabolismo , Pinocitose/fisiologia , Animais , Membrana Celular/efeitos dos fármacos , Células Cultivadas , Cromonas/farmacologia , Inibidores Enzimáticos/farmacologia , Células HEK293 , Humanos , Macrófagos/citologia , Macrófagos/metabolismo , Macrófagos/ultraestrutura , Camundongos , Microscopia Eletrônica de Varredura , Morfolinas/farmacologia , Fosfatidilinositóis/metabolismo , Pinocitose/efeitos dos fármacos , Células RAW 264.7
9.
Int J Mol Sci ; 22(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34360998

RESUMO

Candida albicans, an opportunistic fungus, causes dental caries and contributes to mucosal bacterial dysbiosis leading to a second infection. Furthermore, C.albicans forms biofilms that are resistant to medicinal treatment. To make matters worse, antifungal resistance has spread (albeit slowly) in this species. Thus, it has been imperative to develop novel, antifungal drug compounds. Herein, a peptide was engineered with the sequence of RRFSFWFSFRR-NH2; this was named P19. This novel peptide has been observed to exert disruptive effects on fungal cell membrane physiology. Our results showed that P19 displayed high binding affinity to lipopolysaccharides (LPS), lipoteichoic acids (LTA) and the plasma membrane phosphatidylinositol (PI), phosphatidylserine (PS), cardiolipin, and phosphatidylglycerol (PG), further indicating that the molecular mechanism of P19 was not associated with the receptor recognition, but rather related to competitive interaction with the plasma membrane. In addition, compared with fluconazole and amphotericin B, P19 has been shown to have a lower potential for resistance selection than established antifungal agents.


Assuntos
Antifúngicos/farmacologia , Biofilmes/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Oligopeptídeos/farmacologia , Antifúngicos/química , Candida albicans/fisiologia , Cardiolipinas/metabolismo , Membrana Celular/efeitos dos fármacos , Lipopolissacarídeos/metabolismo , Oligopeptídeos/química , Fosfatidilgliceróis/metabolismo , Fosfatidilinositóis/metabolismo , Fosfatidilserinas/metabolismo , Ácidos Teicoicos/metabolismo , Triptofano/química
10.
Nat Commun ; 12(1): 4267, 2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34257291

RESUMO

The lipid composition of organelles acts as a landmark to define membrane identity and specify subcellular function. Phosphoinositides are anionic lipids acting in protein sorting and trafficking at the trans-Golgi network (TGN). In animal cells, sphingolipids control the turnover of phosphoinositides through lipid exchange mechanisms at endoplasmic reticulum/TGN contact sites. In this study, we discover a mechanism for how sphingolipids mediate phosphoinositide homeostasis at the TGN in plant cells. Using multiple approaches, we show that a reduction of the acyl-chain length of sphingolipids results in an increased level of phosphatidylinositol-4-phosphate (PtdIns(4)P or PI4P) at the TGN but not of other lipids usually coupled to PI4P during exchange mechanisms. We show that sphingolipids mediate Phospholipase C (PLC)-driven consumption of PI4P at the TGN rather than local PI4P synthesis and that this mechanism is involved in the polar sorting of the auxin efflux carrier PIN2 at the TGN. Together, our data identify a mode of action of sphingolipids in lipid interplay at the TGN during protein sorting.


Assuntos
Fosfatidilinositóis/metabolismo , Esfingolipídeos/metabolismo , Rede trans-Golgi/metabolismo , Animais , Retículo Endoplasmático/metabolismo , Humanos , Fosfatidilinositóis/genética , Esfingolipídeos/genética , Fosfolipases Tipo C/metabolismo , Rede trans-Golgi/genética
11.
Nat Commun ; 12(1): 4339, 2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34267198

RESUMO

Pleckstrin homology (PH) domains are presumed to bind phosphoinositides (PIPs), but specific interaction with and regulation by PIPs for most PH domain-containing proteins are unclear. Here we employ a single-molecule pulldown assay to study interactions of lipid vesicles with full-length proteins in mammalian whole cell lysates. Of 67 human PH domain-containing proteins initially examined, 36 (54%) are found to have affinity for PIPs with various specificity, the majority of which have not been reported before. Further investigation of ARHGEF3 reveals distinct structural requirements for its binding to PI(4,5)P2 and PI(3,5)P2, and functional relevance of its PI(4,5)P2 binding. We generate a recursive-learning algorithm based on the assay results to analyze the sequences of 242 human PH domains, predicting that 49% of them bind PIPs. Twenty predicted binders and 11 predicted non-binders are assayed, yielding results highly consistent with the prediction. Taken together, our findings reveal unexpected lipid-binding specificity of PH domain-containing proteins.


Assuntos
Fosfatidilinositóis/metabolismo , Domínios de Homologia à Plecstrina , Proteínas/química , Proteínas/metabolismo , Algoritmos , Animais , Sítios de Ligação , Biologia Computacional/métodos , Células HEK293 , Humanos , Camundongos , Microscopia de Fluorescência , Células NIH 3T3 , Fosfatidilinositóis/química , Fosfatidilserinas/química , Fosfatidilserinas/metabolismo , Proteínas/genética , Fatores de Troca de Nucleotídeo Guanina Rho/química , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Sensibilidade e Especificidade , Proteína rhoA de Ligação ao GTP/metabolismo
13.
Biochem Biophys Res Commun ; 568: 110-115, 2021 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-34214875

RESUMO

The phosphoinositides phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P3] and phosphatidylinositol-3,4-bisphosphate [PtdIns(3,4)P2] function as second messengers and have been implicated in cancerogenesis. The signalling events downstream of PtdIns(3,4,5)P3 and PtdIns(3,4)P2 are mediated through a complex network of phosphoinositide binding effector proteins and phosphatases. In this study, we compared the phosphoinositide effector proteins AKT1, TAPP1, TAPP2, VAV1 and P-REX1 and the phosphoinositide phosphatases PTEN, SHIP1 and INPP4B for their binding affinities to PtdIns(3,4,5)P3 and/or PtdIns(3,4)P2 using Surface Plasmon Resonance. Our results demonstrate that all measured proteins except P-REX1 and VAV1 showed high affinity phosphoinositide binding with KD values in the nM to sub-nM range. Within the effector proteins, AKT1 showed the highest affinity for both PtdIns(3,4,5)P3 and PtdIns(3,4)P2. Of the phosphoinositide phosphatases PTEN displayed the highest affinity towards PtdIns(3,4,5)P3 and PtdIns(3,4)P2. The SHIP1 mutant E452K detected in carcinoma patients had a 100-fold increased affinity to PtdIns(3,4)P2 but not to PtdIns(3,4,5)P3 compared to SHIP1 WT. Distinct mutations in phosphoinositide binding proteins like the patient-derived SHIP1E452K mutant may be involved in the upregulation of PI(3,4)P2 -mediated signalling in tumor cells due to phosphoinositide trapping. Our results add further information to the complex hierarchy of phosphoinositide binding proteins helping to elucidate their functional role in cellular signal transduction.


Assuntos
PTEN Fosfo-Hidrolase/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilinositóis/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Sistemas do Segundo Mensageiro , Humanos , Modelos Moleculares , Ligação Proteica , Transdução de Sinais
14.
Biochim Biophys Acta Biomembr ; 1863(11): 183692, 2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34265284

RESUMO

Phosphoinositides are found in low concentration in cellular membranes but perform numerous functions such as signaling, membrane trafficking, protein recruitment and modulation of protein activity. Spatiotemporal regulation by enzymes that phosphorylate and dephosphorylate the inositol ring results in the production of seven distinct and functionally diverse derivatives. Ionization properties of the phosphorylated headgroups of anionic lipids have been shown to impact how they interact with proteins and lipids in the membrane. While the ionization properties of the three bis and one tris phosphorylated forms have been studied in physiologically relevant model membranes, that of the monophosphorylated forms (i.e., phosphatidylinositol-3-phosphate (PI3P), phosphatidylinositol-4-phosphate (PI4P), phosphatidylinositol-5-phosphate (PI5P)) has received less attention. Here, we used 31P MAS NMR to determine the charge of 5 mol% of the monophosphorylated derivatives in pure dioleoylphosphatidylcholine (DOPC) and DOPC/dioleoylphosphatidylethanolamine (DOPE) bilayers as a function of pH. We find that PI3P, PI4P and PI5P each have unique pKa2 values in a DOPC bilayer, and each is reduced in DOPC/DOPE model membranes through the interaction of their headgroups with DOPE according to the electrostatic-hydrogen bond switch model. In this study, using model membranes mimicking the plasma membrane (inner leaflet), Golgi, nuclear membrane, and endosome (outer leaflet), we show that PI3P, PI4P or PI5P maximize their charge at neutral pH. Our results shed light on the electrostatics of the monophosphorylated headgroups of PI3P, PI4P, and PI5P and form the basis of their intracellular functions.


Assuntos
Membrana Celular/química , Fosfatidilinositóis/química , Concentração de Íons de Hidrogênio , Íons , Bicamadas Lipídicas/química , Lipídeos/química , Espectroscopia de Ressonância Magnética/métodos , Membranas Artificiais
15.
Anal Chem ; 93(27): 9583-9592, 2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34191474

RESUMO

Phosphoinositides (PIPx) play central roles in membrane dynamics and signal transduction of key functions like cellular growth, proliferation, differentiation, migration, and adhesion. They are highly regulated through a network of distinct phosphatidylinositol phosphates consisting of seven groups and three regioisomers in two groups (PIP and PIP2), which arise from phosphorylation at 3', 4', and 5' positions of the inositol ring. Numerous studies have revealed the importance of both fatty acyl chains, degree of phosphorylation, and phosphorylation positions under physiological and pathological states. However, a comprehensive analytical method that allows differentiation of all regioisomeric forms with different acyl side chains and degrees of phosphorylation is still lacking. Here, we present an integrated comprehensive workflow of PIPx analysis utilizing a chiral polysaccharide stationary phase coupled with electrospray ionization high-resolution mass spectrometry with a data independent acquisition technique using the SWATH technology. Correspondingly, a targeted data mining strategy in the untargeted comprehensively acquired MS and MS/MS data was developed. This powerful highly selective method gives a full picture of PIPx profiles in biological samples. Herein, we present for the first time the full PIPx profiles of NIST SRM1950 plasma, Pichia pastoris lipid extract, and HeLa cell extract, including profile changes upon treatment with potential PI3K inhibitor wortmannin. We also illustrate using this inhibitor that measurements of the PIPx profile averaged over the distinct regioisomers by analytical procedures, which cannot differentiate between the individual PIPx isomers, can easily lead to biased conclusions.


Assuntos
Lipidômica , Fosfatidilinositóis/análise , Cromatografia Líquida , Células HeLa , Humanos , Fosfatidilinositol 3-Quinases , Saccharomycetales , Espectrometria de Massas em Tandem
16.
Methods Enzymol ; 652: 49-79, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34059290

RESUMO

Ion channel are embedded in the lipid bilayers of biological membranes. Membrane phospholipids constitute a barrier to ion movement, and they have been considered for a long time as a passive environment for channel proteins. Membrane phospholipids, however, do not only serve as a passive amphipathic environment, but they also modulate channel activity by direct specific lipid-protein interactions. Phosphoinositides are quantitatively minor components of biological membranes, and they play roles in many cellular functions, including membrane traffic, cellular signaling and cytoskeletal organization. Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is mainly found in the inner leaflet of the plasma membrane. Its role as a potential ion channel regulator was first appreciated over two decades ago and by now this lipid is a well-established cofactor or regulator of many different ion channels. The past two decades witnessed the steady development of techniques to study ion channel regulation by phosphoinositides with progress culminating in recent cryoEM structures that allowed visualization of how PI(4,5)P2 opens some ion channels. This chapter will provide an overview of the methods to study regulation by phosphoinositides, focusing on plasma membrane ion channels and PI(4,5)P2.


Assuntos
Fosfatidilinositol 4,5-Difosfato , Fosfatidilinositóis , Membrana Celular , Canais Iônicos , Bicamadas Lipídicas
17.
Int J Mol Sci ; 22(10)2021 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-34069162

RESUMO

Therapeutic elevation of high-density lipoprotein (HDL) is thought to minimize atherogenesis in subjects with dyslipidemia. However, this is not the case in clinical practice. The function of HDL is not determined by its concentration in the plasma but by its specific structural components. We previously identified an index for the prediction of HDL functionality, relative HDL (rHDL) index, and preliminarily explored that dysfunctional HDL (rHDL index value > 2) failed to rescue the damage to endothelial progenitor cells (EPCs). To confirm the effectiveness of the rHDL index for predicting HDL functions, here we evaluated the effects of HDL from patients with different rHDL index values on the endothelial-mesenchymal transition (EndoMT) of EPCs. We also analyzed the lipid species in HDL with different rHDL index values and investigated the structural differences that affect HDL functions. The results indicate that HDL from healthy adults and subjects with an rHDL index value < 2 protected transforming growth factor (TGF)-ß1-stimulated EndoMT by modulating Smad2/3 and Snail activation. HDL from subjects with an rHDL index value > 2 failed to restore the functionality of TGF-ß1-treated EPCs. Lipidomic analysis demonstrated that HDL with different rHDL index values may differ in the composition of triglycerides, phosphatidylcholine, and phosphatidylinositol. In conclusion, we confirmed the applicability of the rHDL index value to predict HDL function and found structural differences that may affect the function of HDL, which warrants further in-depth studies.


Assuntos
Células Progenitoras Endoteliais/metabolismo , Lipoproteínas HDL/química , Lipoproteínas HDL/metabolismo , Idoso , Dislipidemias/sangue , Células Progenitoras Endoteliais/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Lipoproteínas HDL/farmacologia , Masculino , Pessoa de Meia-Idade , Fosfatidilcolinas/sangue , Fosfatidilcolinas/química , Fosfatidilinositóis/sangue , Fosfatidilinositóis/química , Proteínas Smad/metabolismo , Fatores de Transcrição da Família Snail/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Triglicerídeos/sangue , Triglicerídeos/química , Adulto Jovem
18.
Methods Enzymol ; 654: 85-114, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34120726

RESUMO

Voltage sensing phosphatase (VSP), consists of a voltage sensor domain (VSD) like that found in voltage-gated ion channels and a phosphoinositide (PIP) phosphatase region exhibiting remarkable structural similarity to a tumor suppressor enzyme, PTEN. Membrane depolarization activates the enzyme activity through tight coupling between the VSD and enzyme region. The VSD of VSP has a unique nature; it is a self-contained module that can be transferred to other proteins, conferring voltage sensitivity. Thanks to this nature, numerous versions of gene-encoded voltage indicators (GEVIs) have been developed through combination of a fluorescent protein with the VSD of VSP. In addition, VSP itself can also serve as a tool to alter PIP levels in cells. Cellular levels of PIPs, PI(4,5)P2 in particular, can be acutely and transiently reduced using a simple voltage protocol after heterologous expression of VSP. Recent progress in our understanding of the molecular structure and mechanisms underlying VSP facilitates optimization of its molecular properties for its use as a molecular tool.


Assuntos
Fosfatidilinositóis , Monoéster Fosfórico Hidrolases , Monoéster Fosfórico Hidrolases/genética
19.
J Cell Sci ; 134(14)2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34109410

RESUMO

Macropinocytosis allows cells to take up extracellular material in a non-selective manner into large vesicles called macropinosomes. After internalization, macropinosomes acquire phosphatidylinositol 3-phosphate (PtdIns3P) on their limiting membrane as they mature into endosomal-like vesicles. The molecular mechanisms that underlie recycling of membranes and transmembrane proteins from these macropinosomes still need to be defined. Here, we report that JIP4 (officially known as SPAG9), a protein previously described to bind to microtubule motors, is recruited to tubulating subdomains on macropinosomes by the PtdIns3P-binding protein Phafin2 (officially known as PLEKHF2). These JIP4-positive tubulating subdomains on macropinosomes contain F-actin, the retromer recycling complex and the retromer cargo VAMP3. Disruption of the JIP4-Phafin2 interaction, deletion of Phafin2 or inhibition of PtdIns3P production by VPS34 impairs JIP4 recruitment to macropinosomes. Whereas knockout of JIP4 suppresses tubulation, its overexpression enhances tubulation from macropinosomes. JIP4-knockout cells display increased retention of macropinocytic cargo in both early and late macropinosomes. Collectively, these data identify JIP4 and Phafin2 as components of a tubular recycling pathway that operates from macropinosomes. This article has an associated First Person interview with the first author of the paper.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte , Fosfatidilinositóis , Proteínas de Transporte Vesicular , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Endossomos/metabolismo , Humanos , Fosfatidilinositóis/metabolismo , Pinocitose , Ligação Proteica , Transporte Proteico , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo
20.
Artigo em Inglês | MEDLINE | ID: mdl-34111527

RESUMO

Phosphatidylinositol is the parent lipid for the synthesis of seven phosphorylated inositol lipids and each of them play specific roles in numerous processes including receptor-mediated signalling, actin cytoskeleton dynamics and membrane trafficking. PI synthesis is localised to the endoplasmic reticulum (ER) whilst its phosphorylated derivatives are found in other organelles where the lipid kinases also reside. Phosphorylation of PI to phosphatidylinositol (4,5) bisphosphate (PI(4,5)P2) at the plasma membrane and to phosphatidylinositol 4-phosphate (PI4P) at the Golgi are key events in lipid signalling and Golgi function respectively. Here we review a family of proteins, phosphatidylinositol transfer proteins (PITPs), that can mobilise PI from the ER to provide the substrate to the resident kinases for phosphorylation. Recent studies identify specific and overlapping functions for the three soluble PITPs (PITPα, PITPß and PITPNC1) in phospholipase C signalling, neuronal function, membrane trafficking, viral replication and in cancer metastases.


Assuntos
Fosfatidilinositóis/metabolismo , Transporte Biológico , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Transdução de Sinais
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