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1.
Nat Commun ; 12(1): 508, 2021 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-33479206

RESUMO

Thousands of human small and alternative open reading frames (smORFs and alt-ORFs, respectively) have recently been annotated. Many alt-ORFs are co-encoded with canonical proteins in multicistronic configurations, but few of their functions are known. Here, we report the detection of alt-RPL36, a protein co-encoded with human RPL36. Alt-RPL36 partially localizes to the endoplasmic reticulum, where it interacts with TMEM24, which transports the phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) precursor phosphatidylinositol from the endoplasmic reticulum to the plasma membrane. Knock-out of alt-RPL36 increases plasma membrane PI(4,5)P2 levels, upregulates PI3K-AKT-mTOR signaling, and increases cell size. Alt-RPL36 contains four phosphoserine residues, point mutations of which abolish interaction with TMEM24 and, consequently, alt-RPL36 effects on PI3K signaling and cell size. These results implicate alt-RPL36 as an upstream regulator of PI3K-AKT-mTOR signaling. More broadly, the RPL36 transcript encodes two sequence-independent polypeptides that co-regulate translation via different molecular mechanisms, expanding our knowledge of multicistronic human gene functions.


Assuntos
Proteínas de Membrana/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Ribossômicas/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Processamento Alternativo , Sequência de Aminoácidos , Sequência de Bases , Transporte Biológico , Membrana Celular/metabolismo , Regulação para Baixo , Retículo Endoplasmático/metabolismo , Células HEK293 , Humanos , Proteínas de Membrana/genética , Mutação , Fosfatidilinositol 4,5-Difosfato/metabolismo , Ligação Proteica , Proteínas Ribossômicas/genética
2.
Proc Natl Acad Sci U S A ; 118(1)2021 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-33443156

RESUMO

Fertility relies upon pulsatile release of gonadotropin-releasing hormone (GnRH) that drives pulsatile luteinizing hormone secretion. Kisspeptin (KP) neurons in the arcuate nucleus are at the center of the GnRH pulse generation and the steroid feedback control of GnRH secretion. However, KP evokes a long-lasting response in GnRH neurons that is hard to reconcile with periodic GnRH activity required to drive GnRH pulses. Using calcium imaging, we show that 1) the tetrodotoxin-insensitive calcium response evoked by KP relies upon the ongoing activity of canonical transient receptor potential channels maintaining voltage-gated calcium channels in an activated state, 2) the duration of the calcium response is determined by the rate of resynthesis of phosphatidylinositol 4,5-bisphosphate (PIP2), and 3) nitric oxide terminates the calcium response by facilitating the resynthesis of PIP2 via the canonical pathway guanylyl cyclase/3',5'-cyclic guanosine monophosphate/protein kinase G. In addition, our data indicate that exposure to nitric oxide after KP facilitates the calcium response to a subsequent KP application. This effect was replicated using electrophysiology on GnRH neurons in acute brain slices. The interplay between KP and nitric oxide signaling provides a mechanism for modulation of the refractory period of GnRH neurons after KP exposure and places nitric oxide as an important component for tonic GnRH neuronal pulses.


Assuntos
Sinalização do Cálcio/fisiologia , Hormônio Liberador de Gonadotropina/metabolismo , Neurônios/metabolismo , Animais , Núcleo Arqueado do Hipotálamo/metabolismo , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Feminino , Hipotálamo/metabolismo , Kisspeptinas/metabolismo , Hormônio Luteinizante/metabolismo , Masculino , Camundongos , Óxido Nítrico/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatidilinositol 4,5-Difosfato/fisiologia , Cultura Primária de Células/métodos
3.
Methods Mol Biol ; 2251: 91-104, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33481233

RESUMO

Phosphoinositides make up only a small fraction of cellular phospholipids yet control cell function in a fundamental manner. Through protein interactions, phosphoinositides define cellular organelle identity and regulate protein function and organization and recruitment at the cytosol-membrane interface. As a result, perturbations on phosphoinositide metabolism alter cell physiology and lead to a wide range of human diseases, including cancer and diabetes. Among seven phosphoinositide members, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2, also known as PI(4,5)P2 or PIP2) is abundant in the plasma membrane. Besides its role in the second messenger pathway of phospholipase C that cleaves PtdIns(4,5)P2 to form diacylglycerol and inositol-1,4,5-trisphosphate (IP3), PtdIns(4,5)P2 regulates membrane trafficking and the function of the cytoskeleton, ion channels, and transporters. The nanoscale organization of PtdIns(4,5)P2 in the plasma membrane becomes essential to understand cellular signaling specificity in time and space. Here, we describe a single-molecule method to visualize the nanoscale distribution of PtdIns(4,5)P2 in the plasma membrane by using super-resolution microscopy and the dual-color fluorescent probes based on the PLCδ1 pleckstrin homology (PH) domain. This approach can be extended to image other phosphoinositides by changing the specific probes.


Assuntos
Membrana Celular/química , Fosfatidilinositóis/análise , Imagem Individual de Molécula/métodos , Animais , Técnicas de Cultura de Células/métodos , Membrana Celular/metabolismo , Humanos , Membranas/metabolismo , Microscopia de Fluorescência/métodos , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilinositóis/química , Fosfatidilinositóis/metabolismo , Transporte Proteico/fisiologia , Fosfolipases Tipo C/análise , Fosfolipases Tipo C/química , Fosfolipases Tipo C/metabolismo
4.
Proc Natl Acad Sci U S A ; 117(34): 20378-20389, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32764146

RESUMO

The phosphoinositide, phosphatidylinositol 4,5-bisphosphate (PIP2), has long been established as a major contributor to intracellular signaling, primarily by virtue of its role as a substrate for phospholipase C (PLC). Signaling by Gq-protein-coupled receptors triggers PLC-mediated hydrolysis of PIP2 into inositol 1,4,5-trisphosphate and diacylglycerol, which are well known to modulate vascular ion channel activity. Often overlooked, however, is the role PIP2 itself plays in this regulation. Although numerous reports have demonstrated that PIP2 is critical for ion channel regulation, how it impacts vascular function has received scant attention. In this review, we focus on PIP2 as a regulator of ion channels in smooth muscle cells and endothelial cells-the two major classes of vascular cells. We further address the concerted effects of such regulation on vascular function and blood flow control. We close with a consideration of current knowledge regarding disruption of PIP2 regulation of vascular ion channels in disease.


Assuntos
Células Endoteliais/metabolismo , Canais Iônicos/metabolismo , Miócitos de Músculo Liso/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Animais , Endotélio Vascular/metabolismo , Humanos , Músculo Liso Vascular/metabolismo , Doenças Vasculares/metabolismo
5.
Med Hypotheses ; 143: 110148, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32759016

RESUMO

Estrogen hormone acts as a potential key player in providing immunity against certain viral infection. It is found to be associated in providing immunity against acute lungs inflammation and influenza virus by modulating cytokines storm and mediating adaptive immune alterations respectively. Women are less affected by SARS-CoV-2 infection because of the possible influence of estrogen hormone as compared to men. We hypothesized that SARS-CoV-2 causes stress in endoplasmic reticulum (ER) which in turn aggravates the infection, estrogen hormone might play key role in decreasing ER stress by activating estrogen mediated signaling pathways, results in unfolded protein response (UPR). Estrogen governs degradation of phosphotidylinositol 4,5-bisphosphate (PIP2) into diacylglycerol (DAG) and inositol triphosphate (IP3) with the help of phospholipase C. IP3 start in-fluxing Ca+2 ions that helps in UPR activation. To support our hypothesis, we analyzed the data of 162,392 COVID-19 patients to determine the relation of this disease with gender. We observed that 26% of women and 74% of men were affected by SARS-CoV-2. It indicated that women are less affected because of the possible influence of estrogen hormone in women.


Assuntos
Betacoronavirus , Infecções por Coronavirus/fisiopatologia , Estresse do Retículo Endoplasmático/fisiologia , Estrogênios/fisiologia , Modelos Biológicos , Pandemias , Pneumonia Viral/fisiopatologia , Adulto , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/metabolismo , Conjuntos de Dados como Assunto/estatística & dados numéricos , Diglicerídeos/metabolismo , Resistência à Doença , Feminino , Humanos , Inositol 1,4,5-Trifosfato/metabolismo , Masculino , Pessoa de Meia-Idade , Paquistão/epidemiologia , Fosfatidilinositol 4,5-Difosfato/metabolismo , Pneumonia Viral/epidemiologia , Pneumonia Viral/metabolismo , Caracteres Sexuais , Distribuição por Sexo , Transdução de Sinais , Fosfolipases Tipo C/metabolismo , Resposta a Proteínas não Dobradas , Proteínas Virais/biossíntese , Proteínas Virais/genética
6.
PLoS One ; 15(7): e0236201, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32687545

RESUMO

Interaction of phospholipase D2 (PLD2) with phosphatidylinositol (4,5)-bisphosphate (PIP2) is regarded as the critical step of numerous physiological processes. Here we build a full-length model of human PLD2 (hPLD2) combining template-based and ab initio modeling techniques and use microsecond all-atom molecular dynamics (MD) simulations of the protein in contact with a complex membrane to determine hPLD2-PIP2 interactions. MD simulations reveal that the intermolecular interactions preferentially occur between specific PIP2 phosphate groups and hPLD2 residues; the most strongly interacting residues are arginine at the pbox consensus sequence (PX) and pleckstrin homology (PH) domain. Interaction networks indicate formation of clusters at the protein-membrane interface consisting of amino acids, PIP2, and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidic acid (POPA); the largest cluster was in the PH domain.


Assuntos
Membrana Celular/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfolipase D/metabolismo , Sequência de Aminoácidos , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/ultraestrutura , Proteínas de Bactérias/química , Proteínas de Bactérias/ultraestrutura , Sítios de Ligação , Membrana Celular/química , Sequência Consenso , Cristalografia por Raios X , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ácidos Fosfatídicos/metabolismo , Fosfatidilinositol 4,5-Difosfato/química , Fosfolipase D/química , Fosfolipase D/ultraestrutura , Ligação Proteica , Domínios Proteicos , Homologia de Sequência de Aminoácidos
7.
J Virol ; 94(14)2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32376619

RESUMO

HIV-1 assembly occurs principally at the plasma membrane (PM) of infected cells. Gag polyprotein precursors (Pr55Gag) are targeted to the PM, and their binding is mediated by the interaction of myristoylated matrix domain and a PM-specific phosphoinositide, the phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2]. The major synthesis pathway of PI(4,5)P2 involves the activity of phosphatidylinositol-4-phosphate 5-kinase family type 1 composed of three isoforms (PIP5K1α, PIP5K1ß, and PIP5K1γ). To examine whether the activity of a specific PIP5K1 isoform determines proper Pr55Gag localization at the PM, we compared the cellular behavior of Pr55Gag in the context of PIP5K1 inhibition using siRNAs that individually targeted each of the three isoforms in TZM-bl HeLa cells. We found that downregulation of PIP5K1α and PIP5K1γ strongly impaired the targeting of Pr55Gag to the PM with a rerouting of the polyprotein within intracellular compartments. The efficiency of Pr55Gag release was thus impaired through the silencing of these two isoforms, while PIP5K1ß is dispensable for Pr55Gag targeting to the PM. The PM mistargeting due to the silencing of PIP5K1α leads to Pr55Gag hydrolysis through lysosome and proteasome pathways, while the silencing of PIP5K1γ leads to Pr55Gag accumulation in late endosomes. Our findings demonstrated that, within the PIP5K1 family, only the PI(4,5)P2 pools produced by PIP5K1α and PIP5K1γ are involved in the Pr55Gag PM targeting process.IMPORTANCE PM specificity of Pr55Gag membrane binding is mediated through the interaction of PI(4,5)P2 with the matrix (MA) basic residues. It was shown that overexpression of a PI(4,5)P2-depleting enzyme strongly impaired PM localization of Pr55Gag However, cellular factors that control PI(4,5)P2 production required for Pr55Gag-PM targeting have not yet been characterized. In this study, by individually inhibiting PIP5K1 isoforms, we elucidated a correlation between PI(4,5)P2 metabolism pathways mediated by PIP5K1 isoforms and the targeting of Pr55Gag to the PM of TZM-bl HeLa cells. Confocal microscopy analyses of cells depleted from PIP5K1α and PIP5K1γ show a rerouting of Pr55Gag to various intracellular compartments. Notably, Pr55Gag is degraded by the proteasome and/or by the lysosomes in PIP5K1α-depleted cells, while Pr55Gag is targeted to endosomal vesicles in PIP5K1γ-depleted cells. Thus, our results highlight, for the first time, the roles of PIP5K1α and PIP5K1γ as determinants of Pr55Gag targeting to the PM.


Assuntos
Membrana Celular/metabolismo , Regulação para Baixo , Regulação Enzimológica da Expressão Gênica , HIV-1/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/biossíntese , Precursores de Proteínas/metabolismo , Membrana Celular/genética , Membrana Celular/virologia , Endossomos/genética , Endossomos/metabolismo , Endossomos/virologia , HIV-1/genética , Células HeLa , Humanos , Lisossomos/genética , Lisossomos/metabolismo , Lisossomos/virologia , Fosfatidilinositol 4,5-Difosfato/genética , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Precursores de Proteínas/genética , Proteólise
8.
Soft Matter ; 16(21): 4941-4954, 2020 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-32436537

RESUMO

The dynamics and organization of the actin cytoskeleton are crucial to many cellular events such as motility, polarization, cell shaping, and cell division. The intracellular and extracellular signaling associated with this cytoskeletal network is communicated through cell membranes. Hence the organization of membrane macromolecules and actin filament assembly are highly interdependent. Although the actin-membrane linkage is known to happen through many routes, the major class of interactions is through the direct interaction of actin-binding proteins with the lipid class containing poly-phosphatidylinositols (PPIs). Among the PPIs, phosphatidylinositol bisphosphate (PI(4,5)P2) acts as a significant factor controlling actin polymerization in the proximity of the membrane by binding to actin-associated proteins. The molecular interactions between these actin-binding proteins and the membrane lipids remain elusive. Here, using molecular modeling, analytical theory, and experimental methods, we investigate the binding of three different actin-binding proteins, mDia2, NWASP, and gelsolin, to membranes containing PI(4,5)P2 lipids. We perform molecular dynamics simulations on the protein-bilayer system and analyze the membrane binding in the form of hydrogen bonds and salt bridges at various PI(4,5)P2 and cholesterol concentrations. Our experimental study with PI(4,5)P2-containing large unilamellar vesicles mimics the computational experiments. Using the multivalencies of the proteins obtained in molecular simulations and the cooperative binding mechanisms of the proteins, we also propose a multivalent binding model that predicts the actin filament distributions at various PI(4,5)P2 and protein concentrations.


Assuntos
Gelsolina/química , Bicamadas Lipídicas/química , Proteínas Associadas aos Microtúbulos/química , Simulação de Dinâmica Molecular , NADPH Desidrogenase/química , Fosfatidilinositol 4,5-Difosfato/química , Animais , Membrana Celular/química , Membrana Celular/metabolismo , Gelsolina/metabolismo , Bicamadas Lipídicas/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , NADPH Desidrogenase/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Ligação Proteica
9.
Proc Natl Acad Sci U S A ; 117(24): 13468-13479, 2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32467162

RESUMO

The functions of nervous and neuroendocrine systems rely on fast and tightly regulated release of neurotransmitters stored in secretory vesicles through SNARE-mediated exocytosis. Few proteins, including tomosyn (STXBP5) and amisyn (STXBP6), were proposed to negatively regulate exocytosis. Little is known about amisyn, a 24-kDa brain-enriched protein with a SNARE motif. We report here that full-length amisyn forms a stable SNARE complex with syntaxin-1 and SNAP-25 through its C-terminal SNARE motif and competes with synaptobrevin-2/VAMP2 for the SNARE-complex assembly. Furthermore, amisyn contains an N-terminal pleckstrin homology domain that mediates its transient association with the plasma membrane of neurosecretory cells by binding to phospholipid PI(4,5)P2 However, unlike synaptrobrevin-2, the SNARE motif of amisyn is not sufficient to account for the role of amisyn in exocytosis: Both the pleckstrin homology domain and the SNARE motif are needed for its inhibitory function. Mechanistically, amisyn interferes with the priming of secretory vesicles and the sizes of releasable vesicle pools, but not vesicle fusion properties. Our biochemical and functional analyses of this vertebrate-specific protein unveil key aspects of negative regulation of exocytosis.


Assuntos
Exocitose , Fosfatidilinositol 4,5-Difosfato/metabolismo , Proteína 2 Associada à Membrana da Vesícula/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Membrana Celular/metabolismo , Células Cultivadas , Células Cromafins/metabolismo , Humanos , Lipossomos/metabolismo , Fusão de Membrana , Células PC12 , Domínios de Homologia à Plecstrina , Ligação Proteica , Ratos , Proteínas SNARE/metabolismo , Proteína 25 Associada a Sinaptossoma/metabolismo , Sintaxina 1/metabolismo , Vertebrados , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genética
10.
PLoS Comput Biol ; 16(4): e1007777, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32271757

RESUMO

The co-localization of Cluster-of-Differentiation-44 protein (CD44) and cytoplasmic adaptors in specific membrane environments is crucial for cell adhesion and migration. The process is controlled by two different pathways: On the one hand palmitoylation keeps CD44 in lipid raft domains and disables the linking to the cytoplasmic adaptor, whereas on the other hand, the presence of phosphatidylinositol-4,5-biphosphate (PIP2) lipids accelerates the formation of the CD44-adaptor complex. The molecular mechanism explaining how CD44 is migrating into and out of the lipid raft domains and its dependence on both palmitoylations and the presence of PIP2 remains, however, elusive. In this study, we performed extensive molecular dynamics simulations to study the raft affinity and translocation of CD44 in phase separated model membranes as well as more realistic plasma membrane environments. We observe a delicate balance between the influence of the palmitoylations and the presence of PIP2 lipids: whereas the palmitoylations of CD44 increases the affinity for raft domains, PIP2 lipids have the opposite effect. Additionally, we studied the association between CD44 and the membrane adaptor FERM in dependence of these factors. We find that the presence of PIP2 lipids allows CD44 and FERM to associate in an experimentally observed binding mode whereas the highly palmitoylated species shows no binding affinity. Together, our results shed light on the sophisticated mechanism on how membrane translocation and peripheral protein association can be controlled by both protein modifications and membrane composition.


Assuntos
Receptores de Hialuronatos , Lipoilação/fisiologia , Microdomínios da Membrana , Simulação de Dinâmica Molecular , Fosfatidilinositol 4,5-Difosfato , Membrana Celular/química , Membrana Celular/metabolismo , Biologia Computacional , Citoplasma/química , Citoplasma/metabolismo , Receptores de Hialuronatos/química , Receptores de Hialuronatos/metabolismo , Microdomínios da Membrana/química , Microdomínios da Membrana/metabolismo , Fosfatidilinositol 4,5-Difosfato/química , Fosfatidilinositol 4,5-Difosfato/metabolismo
11.
Arterioscler Thromb Vasc Biol ; 40(5): 1311-1324, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32188273

RESUMO

OBJECTIVE: TMEM55B (transmembrane protein 55B) is a phosphatidylinositol-(4,5)-bisphosphate (PI[4,5]P2) phosphatase that regulates cellular cholesterol, modulates LDLR (low-density lipoprotein receptor) decay, and lysosome function. We tested the effects of Tmem55b knockdown on plasma lipids in mice and assessed the roles of LDLR lysosomal degradation and change in (PI[4,5]P2) in mediating these effects. Approach and Results: Western diet-fed C57BL/6J mice were treated with antisense oligonucleotides against Tmem55b or a nontargeting control for 3 to 4 weeks. Hepatic Tmem55b transcript and protein levels were reduced by ≈70%, and plasma non-HDL (high-density lipoprotein) cholesterol was increased ≈1.8-fold (P<0.0001). Immunoblot analysis of fast protein liquid chromatography (FPLC) fractions revealed enrichment of ApoE-containing particles in the LDL size range. In contrast, Tmem55b knockdown had no effect on plasma cholesterol in Ldlr-/- mice. In primary hepatocytes and liver tissues from Tmem55b knockdown mice, there was decreased LDLR protein. In the hepatocytes, there was increased lysosome staining and increased LDLR-lysosome colocalization. Impairment of lysosome function (incubation with NH4Cl or knockdown of the lysosomal proteins LAMP1 or RAB7) abolished the effect of TMEM55B knockdown on LDLR in HepG2 (human hepatoma) cells. Colocalization of the recycling endosome marker RAB11 (Ras-related protein 11) with LDLR in HepG2 cells was reduced by 50% upon TMEM55B knockdown. Finally, knockdown increased hepatic PI(4,5)P2 levels in vivo and in HepG2 cells, while TMEM55B overexpression in vitro decreased PI(4,5)P2. TMEM55B knockdown decreased, whereas overexpression increased, LDL uptake in HepG2 cells. Notably, the TMEM55B overexpression effect was reversed by incubation with PI(4,5)P2. Conclusions: These findings indicate a role for TMEM55B in regulating plasma cholesterol levels by affecting PI(4,5)P2-mediated LDLR lysosomal degradation.


Assuntos
Colesterol/sangue , Hepatócitos/metabolismo , Fígado/metabolismo , Lisossomos/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatases de Fosfoinositídeos/metabolismo , Receptores de LDL/metabolismo , Animais , Dieta Hiperlipídica , Regulação para Baixo , Feminino , Células Hep G2 , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfatases de Fosfoinositídeos/genética , Transporte Proteico , Proteólise , Receptores de LDL/genética , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo
12.
J Cell Biol ; 219(3)2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-32211893

RESUMO

The polyphosphoinositides (PPIn) are central regulatory lipids that direct membrane function in eukaryotic cells. Understanding how their synthesis is regulated is crucial to revealing these lipids' role in health and disease. PPIn are derived from the major structural lipid, phosphatidylinositol (PI). However, although the distribution of most PPIn has been characterized, the subcellular localization of PI available for PPIn synthesis is not known. Here, we used several orthogonal approaches to map the subcellular distribution of PI, including localizing exogenous fluorescent PI, as well as detecting lipid conversion products of endogenous PI after acute chemogenetic activation of PI-specific phospholipase and 4-kinase. We report that PI is broadly distributed throughout intracellular membrane compartments. However, there is a surprising lack of PI in the plasma membrane compared with the PPIn. These experiments implicate regulation of PI supply to the plasma membrane, as opposed to regulation of PPIn-kinases, as crucial to the control of PPIn synthesis and function at the PM.


Assuntos
Membrana Celular/metabolismo , Membranas Intracelulares/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilinositóis/metabolismo , Animais , Células COS , Chlorocebus aethiops , Diglicerídeos/metabolismo , Cinética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/metabolismo
13.
Proc Natl Acad Sci U S A ; 117(14): 7803-7813, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32213593

RESUMO

Protein-lipid interactions are a key element of the function of many integral membrane proteins. These potential interactions should be considered alongside the complexity and diversity of membrane lipid composition. Inward rectifier potassium channel (Kir) Kir2.2 has multiple interactions with plasma membrane lipids: Phosphatidylinositol (4, 5)-bisphosphate (PIP2) activates the channel; a secondary anionic lipid site has been identified, which augments the activation by PIP2; and cholesterol inhibits the channel. Molecular dynamics simulations are used to characterize in molecular detail the protein-lipid interactions of Kir2.2 in a model of the complex plasma membrane. Kir2.2 has been simulated with multiple, functionally important lipid species. From our simulations we show that PIP2 interacts most tightly at the crystallographic interaction sites, outcompeting other lipid species at this site. Phosphatidylserine (PS) interacts at the previously identified secondary anionic lipid interaction site, in a PIP2 concentration-dependent manner. There is interplay between these anionic lipids: PS interactions are diminished when PIP2 is not present in the membrane, underlining the need to consider multiple lipid species when investigating protein-lipid interactions.


Assuntos
Metabolismo dos Lipídeos/genética , Lipídeos/genética , Canais de Potássio Corretores do Fluxo de Internalização/genética , Animais , Ânions/metabolismo , Membrana Celular/genética , Membrana Celular/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Simulação de Dinâmica Molecular , Fosfatidilinositol 4,5-Difosfato/metabolismo , Potássio/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo
14.
Nat Chem Biol ; 16(4): 400-407, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32198492

RESUMO

The signal transduction enzyme phospholipase D1 (PLD1) hydrolyzes phosphatidylcholine to generate the lipid second-messenger phosphatidic acid, which plays roles in disease processes such as thrombosis and cancer. PLD1 is directly and synergistically regulated by protein kinase C, Arf and Rho GTPases, and the membrane lipid phosphatidylinositol-4,5-bisphosphate (PIP2). Here, we present a 1.8 Å-resolution crystal structure of the human PLD1 catalytic domain, which is characterized by a globular fold with a funnel-shaped hydrophobic cavity leading to the active site. Adjacent is a PIP2-binding polybasic pocket at the membrane interface that is essential for activity. The C terminus folds into and contributes part of the catalytic pocket, which harbors a phosphohistidine that mimics an intermediate stage of the catalytic cycle. Mapping of PLD1 mutations that disrupt RhoA activation identifies the RhoA-PLD1 binding interface. This structure sheds light on PLD1 regulation by lipid and protein effectors, enabling rationale inhibitor design for this well-studied therapeutic target.


Assuntos
Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfolipase D/metabolismo , Fosfolipase D/ultraestrutura , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Células COS , Catálise , Domínio Catalítico , Chlorocebus aethiops , Humanos , Lipídeos de Membrana , Fosfatidilcolinas , Ligação Proteica , Proteína Quinase C/metabolismo , Sistemas do Segundo Mensageiro , Transdução de Sinais/efeitos dos fármacos
15.
Int J Mol Sci ; 21(3)2020 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-32050555

RESUMO

Cholesterol is a critical component of eukaryotic membranes, where it contributes to regulating transmembrane signaling, cell-cell interaction, and ion transport. Dysregulation of cholesterol levels in the brain may induce neurodegenerative diseases, such as Alzheimer's disease, Parkinson disease, and Huntington disease. We previously reported that augmenting membrane cholesterol level regulates ion channels by decreasing the level of phosphatidylinositol 4,5-bisphosphate (PIP2), which is closely related to ß-amyloid (Aß) production. In addition, cholesterol enrichment decreased PIP2 levels by increasing the expression of the ß1 isoform of phospholipase C (PLC) in cultured cells. In this study, we examined the effect of a high-cholesterol diet on phospholipase C (PLCß1) expression and PIP2 levels in rat brain. PIP2 levels were decreased in the cerebral cortex in rats on a high-cholesterol diet. Levels of PLCß1 expression correlated with PIP2 levels. However, cholesterol and PIP2 levels were not correlated, suggesting that PIP2 level is regulated by cholesterol via PLCß1 expression in the brain. Thus, there exists cross talk between cholesterol and PIP2 that could contribute to the pathogenesis of neurodegenerative diseases.


Assuntos
Encéfalo/metabolismo , Colesterol/farmacologia , Dieta Hiperlipídica/efeitos adversos , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfolipase C beta/genética , Animais , Colesterol/metabolismo , Masculino , Fosfolipase C beta/metabolismo , Ratos , Ratos Sprague-Dawley
16.
J Cardiovasc Pharmacol Ther ; 25(4): 354-363, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32052660

RESUMO

BACKGROUND: Insulin resistance (IR) is a well-known risk factor for cardiovascular complications. This study aimed to investigate the effect of a dietary model of IR in mice on cardiac remodeling, cardiac ß-arrestin2 signaling, and the protective effects of carvedilol as a ß-arrestin-biased agonist. METHODS AND RESULTS: Insulin resistance was induced by feeding mice high-fructose/high-fat diet (HFrHFD) for 16 weeks. Carvedilol was adiministered for 4 weeks starting at week 13. At the end of the experiment, body weight, heart weight, left and right ventricular thickness, visceral fat weight, fasting blood glucose (FBG), serum insulin, IR index, and serum endothelin-1 were measured. In addition, cardiac tissue samples were histopathologically examined. Also, cardiac levels of cardiotrophin-1, ß-arrestin2, phosphatidylinositol 4,5 bisphosphate (PIP2), diacylglycerol (DAG), and phosphoserine 473 Akt (pS473 Akt) were measured. Results showed significant increases in the FBG, serum insulin, IR index, serum endothelin-1, cardiac DAG, cardiac fibrosis, and degenerated cardiac myofibrils in HFrHFD-fed mice associated with a significant reduction in cardiac levels of cardiotrophin-1, ß-arrestin2, PIP2, and pS473 Akt. On the other hand, carvedilol significantly reduced the heart weight, FBG, serum insulin, IR index, serum endothelin-1, cardiac DAG, left ventricular thickness, right ventricular fibrosis, and degeneration of cardiac myofibrils. In addition, carvedilol significantly increased cardiac levels of cardiotrophin-1, ß-arrestin2, PIP2, and pS473 Akt. CONCLUSION: Carvedilol enhances cardiac ß-arrestin2 signaling and reduces cardiac remodeling in HFrHFD-fed mice.


Assuntos
Cardiomegalia/prevenção & controle , Carvedilol/farmacologia , Resistência à Insulina , Miócitos Cardíacos/efeitos dos fármacos , Função Ventricular Esquerda/efeitos dos fármacos , Função Ventricular Direita/efeitos dos fármacos , beta-Arrestina 2/agonistas , Animais , Cardiomegalia/etiologia , Cardiomegalia/metabolismo , Cardiomegalia/fisiopatologia , Citocinas/metabolismo , Dieta Hiperlipídica , Açúcares da Dieta , Modelos Animais de Doenças , Fibrose , Frutose , Masculino , Camundongos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Remodelação Ventricular/efeitos dos fármacos , beta-Arrestina 2/metabolismo
17.
Int J Mol Sci ; 21(2)2020 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-31936257

RESUMO

Bradykinin (BK), a hormone inducing pain and inflammation, is known to inhibit potassium M-currents (IM) and to increase the excitability of the superior cervical ganglion (SCG) neurons by activating the Ca2+-calmodulin pathway. M-current is also reduced by muscarinic agonists through the depletion of membrane phosphatidylinositol 4,5-biphosphate (PIP2). Similarly, the activation of muscarinic receptors inhibits the current through two-pore domain potassium channels (K2P) of the "Tandem of pore-domains in a Weakly Inward rectifying K+ channel (TWIK)-related channels" (TREK) subfamily by reducing PIP2 in mouse SCG neurons (mSCG). The aim of this work was to test and characterize the modulation of TREK channels by bradykinin. We used the perforated-patch technique to investigate riluzole (RIL) activated currents in voltage- and current-clamp experiments. RIL is a drug used in the palliative treatment of amyotrophic lateral sclerosis and, in addition to blocking voltage-dependent sodium channels, it also selectively activates the K2P channels of the TREK subfamily. A cell-attached patch-clamp was also used to investigate TREK-2 single channel currents. We report here that BK reduces spike frequency adaptation (SFA), inhibits the riluzole-activated current (IRIL), which flows mainly through TREK-2 channels, by about 45%, and reduces the open probability of identified single TREK-2 channels in cultured mSCG cells. The effect of BK on IRIL was precluded by the bradykinin receptor (B2R) antagonist HOE-140 (d-Arg-[Hyp3, Thi5, d-Tic7, Oic8]BK) but also by diC8PIP2 which prevents PIP2 depletion when phospholipase C (PLC) is activated. On the contrary, antagonizing inositol triphosphate receptors (IP3R) using 2-aminoethoxydiphenylborane (2-APB) or inhibiting protein kinase C (PKC) with bisindolylmaleimide did not affect the inhibition of IRIL by BK. In conclusion, bradykinin inhibits TREK-2 channels through the activation of B2Rs resulting in PIP2 depletion, much like we have demonstrated for muscarinic agonists. This mechanism implies that TREK channels must be relevant for the capture of information about pain and visceral inflammation.


Assuntos
Bradicinina/metabolismo , Neurônios/efeitos dos fármacos , Fosfatidilinositol 4,5-Difosfato/metabolismo , Canais de Potássio de Domínios Poros em Tandem/genética , Sistema Nervoso Simpático/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Animais , Bradicinina/administração & dosagem , Bradicinina/análogos & derivados , Bradicinina/genética , Bradicinina/farmacologia , Células Cultivadas , Humanos , Camundongos , Agonistas Muscarínicos/farmacologia , Neurônios/patologia , Técnicas de Patch-Clamp , Fosfatidilinositol 4,5-Difosfato/genética , Potássio/metabolismo , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Receptores Muscarínicos/genética , Riluzol/farmacologia , Bloqueadores dos Canais de Sódio/farmacologia , Gânglio Cervical Superior/efeitos dos fármacos , Sistema Nervoso Simpático/metabolismo , Fosfolipases Tipo C
18.
FASEB J ; 34(2): 2147-2160, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31908005

RESUMO

Profilin is a major regulator of actin dynamics in multiple specific processes localized in different cellular compartments. This specificity is not only meditated by its binding to actin but also its interaction with phospholipids such as phosphatidylinositol (4,5)-bisphosphate (PIP2 ) at the membrane and a plethora of proteins containing poly-L-proline (PLP) stretches. These interactions are fine-tuned by posttranslational modifications such as phosphorylation. Several phospho-sites have already been identified for profilin1, the ubiquitously expressed isoform. However, little is known about the phosphorylation of profilin2a. Profilin2a is a neuronal isoform important for synapse function. Here, we identified several putative profilin2a phospho-sites in silico and tested recombinant phospho-mimetics with regard to their actin-, PLP-, and PIP2 -binding properties. Moreover, we assessed their impact on actin dynamics employing a pyrene-actin polymerization assay. Results indicate that distinct phospho-sites modulate specific profilin2a functions. We could identify a molecular switch site at serine residue 71 which completely abrogated actin binding-as well as other sites important for fine-tuning of different functions, for example, tyrosine 29 for PLP binding. Our findings suggest that differential profilin2a phosphorylation is a sensitive mechanism for regulating its neuronal functions. Moreover, the dysregulation of profilin2a phosphorylation may contribute to neurodegeneration.


Assuntos
Actinas/química , Profilinas/química , Multimerização Proteica , Actinas/metabolismo , Humanos , Neurônios/metabolismo , Fosfatidilinositol 4,5-Difosfato/química , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosforilação , Profilinas/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo
19.
Nat Commun ; 11(1): 231, 2020 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-31932584

RESUMO

The opening of a fusion pore during exocytosis creates the first aqueous connection between the lumen of a vesicle and the extracellular space. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) mediate the formation of these dynamic structures, and their kinetic transitions are tightly regulated by accessory proteins at the synapse. Here, we utilize two single molecule approaches, nanodisc-based planar bilayer electrophysiology and single-molecule FRET, to address the relationship between SNARE complex assembly and rapid (micro-millisecond) fusion pore transitions, and to define the role of accessory proteins. Synaptotagmin (syt) 1, a major Ca2+-sensor for synaptic vesicle exocytosis, drove the formation of an intermediate: committed trans-SNARE complexes that form large, stable pores. Once open, these pores could only be closed by the action of the ATPase, NSF. Time-resolved measurements revealed that NSF-mediated pore closure occurred via a complex 'stuttering' mechanism. This simplified system thus reveals the dynamic formation and dissolution of fusion pores.


Assuntos
Cálcio/metabolismo , Fusão de Membrana , Proteínas Sensíveis a N-Etilmaleimida/metabolismo , Proteínas SNARE/metabolismo , Sinaptotagmina I/metabolismo , Animais , Exocitose , Cinética , Bicamadas Lipídicas/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Multimerização Proteica , Ratos , Proteínas SNARE/química , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida/metabolismo , Vesículas Sinápticas/metabolismo , Sinaptotagmina I/genética
20.
Arch Biochem Biophys ; 682: 108261, 2020 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-31923392

RESUMO

Membrane lipids are key determinants in the regulation of voltage-gated ion channels. Phosphatidylinositol 4,5-bisphosphate (PIP2), a native membrane phospholipid, has been involved in the maintenance of the current amplitude and in the voltage-independent regulation of voltage-gated calcium channels (VGCC). However, the nature of the PIP2 regulation on VGCC has not been fully elucidated. This work aimed to investigate whether the interacting PIP2 electrostatic charges may account for maintaining the current amplitude of CaV2.2 channels. Furthermore, we tested whether charge shielding of PIP2 mimics the voltage-independent inhibition induced by M1 muscarinic acetylcholine receptor (M1R) activation. Therefore, neomycin, a polycation that has been shown to block electrostatic interactions of PIP2, was intracellularly dialyzed in superior cervical ganglion (SCG) neurons of the rat. Consistently, neomycin time-dependently diminished the calcium current amplitude letting the channel exhibit the hallmarks of the voltage-independent regulation. These results support that interacting PIP2 charges not only underly the maintenance of the channel-current but also that charge screening of PIP2 by itself unveils the voltage-independent features of CaV2.2 channels in SCG neurons.


Assuntos
Canais de Cálcio Tipo N/metabolismo , Cálcio/metabolismo , Neomicina/farmacologia , Neurônios/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Sistema Nervoso Simpático/citologia , Animais , Cátions , Masculino , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp , Fosfolipídeos/química , Ratos , Ratos Wistar , Receptores Muscarínicos/metabolismo , Transdução de Sinais , Eletricidade Estática
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