RESUMO
PtdIns and its phosphorylated derivatives, the phosphoinositides, are the biochemical components of a major pathway of intracellular signaling in all eukaryotic cells. These lipids are few in terms of cohort of unique positional isomers, and are quantitatively minor species of the bulk cellular lipidome. Nevertheless, phosphoinositides regulate an impressively diverse set of biological processes. It is from that perspective that perturbations in phosphoinositide-dependent signaling pathways are increasingly being recognized as causal foundations of many human diseases - including cancer. Although phosphatidylinositol transfer proteins (PITPs) are not enzymes, these proteins are physiologically significant regulators of phosphoinositide signaling. As such, PITPs are conserved throughout the eukaryotic kingdom. Their biological importance notwithstanding, PITPs remain understudied. Herein, we review current information regarding PITP biology primarily focusing on how derangements in PITP function disrupt key signaling/developmental pathways and are associated with a growing list of pathologies in mammals.
Assuntos
Neoplasias , Fosfatidilinositóis , Proteínas de Transferência de Fosfolipídeos , Transdução de Sinais , Humanos , Neoplasias/metabolismo , Animais , Proteínas de Transferência de Fosfolipídeos/metabolismo , Proteínas de Transferência de Fosfolipídeos/genética , Fosfatidilinositóis/metabolismoRESUMO
Regulated hydrolysis of the phosphoinositide phosphatidylinositol(4,5)-bis-phosphate to diacylglycerol and inositol-1,4,5-P3 defines a major eukaryotic pathway for translation of extracellular cues to intracellular signaling circuits. Members of the lipid-activated protein kinase C isoenzyme family (PKCs) play central roles in this signaling circuit. One of the regulatory mechanisms employed to downregulate stimulated PKC activity is via a proteasome-dependent degradation pathway that is potentiated by peptidyl-prolyl isomerase Pin1. Here, we show that contrary to prevailing models, Pin1 does not regulate conventional PKC isoforms α and ßII via a canonical cis-trans isomerization of the peptidyl-prolyl bond. Rather, Pin1 acts as a PKC binding partner that controls PKC activity via sequestration of the C-terminal tail of the kinase. The high-resolution structure of full-length Pin1 complexed to the C-terminal tail of PKCßII reveals that a novel bivalent interaction mode underlies the non-catalytic mode of Pin1 action. Specifically, Pin1 adopts a conformation in which it uses the WW and PPIase domains to engage two conserved phosphorylated PKC motifs, the turn motif and hydrophobic motif, respectively. Hydrophobic motif is a non-canonical Pin1-interacting element. The structural information combined with the results of extensive binding studies and experiments in cultured cells suggest that non-catalytic mechanisms represent unappreciated modes of Pin1-mediated regulation of AGC kinases and other key enzymes/substrates.
Assuntos
Peptidilprolil Isomerase de Interação com NIMA , Ligação Proteica , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Peptidilprolil Isomerase de Interação com NIMA/química , Peptidilprolil Isomerase de Interação com NIMA/genética , Humanos , Proteína Quinase C/metabolismo , Proteína Quinase C/química , Proteína Quinase C/genética , Conformação ProteicaRESUMO
Regulated hydrolysis of the phosphoinositide phosphatidylinositol(4,5)-bis-phosphate to diacylglycerol and inositol-1,4,5-P3 defines a major eukaryotic pathway for translation of extracellular cues to intracellular signaling circuits. Members of the lipid-activated protein kinase C isoenzyme family (PKCs) play central roles in this signaling circuit. One of the regulatory mechanisms employed to downregulate stimulated PKC activity is via a proteasome-dependent degradation pathway that is potentiated by peptidyl-prolyl isomerase Pin1. Here, we show that contrary to prevailing models, Pin1 does not regulate conventional PKC isoforms α and ßII via a canonical cis-trans isomerization of the peptidyl-prolyl bond. Rather, Pin1 acts as a PKC binding partner that controls PKC activity via sequestration of the C-terminal tail of the kinase. The high-resolution structure of Pin1 complexed to the C-terminal tail of PKCßII reveals that a novel bivalent interaction mode underlies the non-catalytic mode of Pin1 action. Specifically, Pin1 adopts a compact conformation in which it engages two conserved phosphorylated PKC motifs, the turn motif and hydrophobic motif, the latter being a non-canonical Pin1-interacting element. The structural information, combined with the results of extensive binding studies and in vivo experiments suggest that non-catalytic mechanisms represent unappreciated modes of Pin1-mediated regulation of AGC kinases and other key enzymes/substrates.
RESUMO
Defects in insulin processing and granule maturation are linked to pancreatic beta-cell failure during type 2 diabetes (T2D). Phosphatidylinositol transfer protein alpha (PITPNA) stimulates activity of phosphatidylinositol (PtdIns) 4-OH kinase to produce sufficient PtdIns-4-phosphate (PtdIns-4-P) in the trans-Golgi network to promote insulin granule maturation. PITPNA in beta-cells of T2D human subjects is markedly reduced suggesting its depletion accompanies beta-cell dysfunction. Conditional deletion of Pitpna in the beta-cells of Ins-Cre, Pitpnaflox/flox mice leads to hyperglycemia resulting from decreasing glucose-stimulated insulin secretion (GSIS) and reducing pancreatic beta-cell mass. Furthermore, PITPNA silencing in human islets confirms its role in PtdIns-4-P synthesis and leads to impaired insulin granule maturation and docking, GSIS, and proinsulin processing with evidence of ER stress. Restoration of PITPNA in islets of T2D human subjects reverses these beta-cell defects and identify PITPNA as a critical target linked to beta-cell failure in T2D.
Assuntos
Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , Ilhotas Pancreáticas , Animais , Humanos , Camundongos , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Glucose/metabolismo , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Proinsulina/metabolismoRESUMO
Cellular membranes are critical platforms for intracellular signaling that involve complex interfaces between lipids and proteins, and a web of interactions between a multitude of lipid metabolic pathways. Membrane lipids impart structural and functional information in this regulatory circuit that encompass biophysical parameters such as membrane thickness and fluidity, as well as chaperoning the interactions of protein binding partners. Phosphatidylinositol and its phosphorylated derivatives, the phosphoinositides, play key roles in intracellular membrane signaling, and these involvements are translated into an impressively diverse set of biological outcomes. The phosphatidylinositol transfer proteins (PITPs) are key regulators of phosphoinositide signaling. Found in a diverse array of organisms from plants, yeast and apicomplexan parasites to mammals, PITPs were initially proposed to be simple transporters of lipids between intracellular membranes. It now appears increasingly unlikely that the soluble versions of these proteins perform such functions within the cell. Rather, these serve to facilitate the activity of intrinsically biologically insufficient inositol lipid kinases and, in so doing, promote diversification of the biological outcomes of phosphoinositide signaling. The central engine for execution of such functions is the lipid exchange cycle that is a fundamental property of PITPs. How PITPs execute lipid exchange remains very poorly understood. Molecular dynamics simulation approaches are now providing the first atomistic insights into how PITPs, and potentially other lipid-exchange/transfer proteins, operate.
Assuntos
Inositol/metabolismo , Lipídeos de Membrana/metabolismo , Proteínas de Transferência de Fosfolipídeos/fisiologia , Fosfotransferases/metabolismo , Transdução de Sinais , Animais , HumanosRESUMO
Phosphoinositides are key regulators of a large number of diverse cellular processes that include membrane trafficking, plasma membrane receptor signaling, cell proliferation, and transcription. How a small number of chemically distinct phosphoinositide signals are functionally amplified to exert specific control over such a diverse set of biological outcomes remains incompletely understood. To this end, a novel mechanism is now taking shape, and it involves phosphatidylinositol (PtdIns) transfer proteins (PITPs). The concept that PITPs exert instructive regulation of PtdIns 4-OH kinase activities and thereby channel phosphoinositide production to specific biological outcomes, identifies PITPs as central factors in the diversification of phosphoinositide signaling. There are two evolutionarily distinct families of PITPs: the Sec14-like and the StAR-related lipid transfer domain (START)-like families. Of these two families, the START-like PITPs are the least understood. Herein, we review recent insights into the biochemical, cellular, and physiological function of both PITP families with greater emphasis on the START-like PITPs, and we discuss the underlying mechanisms through which these proteins regulate phosphoinositide signaling and how these actions translate to human health and disease.
Assuntos
Eucariotos/citologia , Eucariotos/metabolismo , Fosfatidilinositóis/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Transdução de Sinais , Animais , Humanos , Proteínas de Transferência de Fosfolipídeos/químicaRESUMO
Phosphatidylinositol-transfer proteins (PITPs) regulate phosphoinositide signaling in eukaryotic cells. The defining feature of PITPs is their ability to exchange phosphatidylinositol (PtdIns) molecules between membranes, and this property is central to PITP-mediated regulation of lipid signaling. However, the details of the PITP-mediated lipid exchange cycle remain entirely obscure. Here, all-atom molecular dynamics simulations of the mammalian StART-like PtdIns/phosphatidylcholine (PtdCho) transfer protein PITPα, both on membrane bilayers and in solvated systems, informed downstream biochemical analyses that tested key aspects of the hypotheses generated by the molecular dynamics simulations. These studies provided five key insights into the PITPα lipid exchange cycle: (i) interaction of PITPα with the membrane is spontaneous and mediated by four specific protein substructures; (ii) the ability of PITPα to initiate closure around the PtdCho ligand is accompanied by loss of flexibility of two helix/loop regions, as well as of the C-terminal helix; (iii) the energy barrier of phospholipid extraction from the membrane is lowered by a network of hydrogen bonds between the lipid molecule and PITPα; (iv) the trajectory of PtdIns or PtdCho into and through the lipid-binding pocket is chaperoned by sets of PITPα residues conserved throughout the StART-like PITP family; and (v) conformational transitions in the C-terminal helix have specific functional involvements in PtdIns transfer activity. Taken together, these findings provide the first mechanistic description of key aspects of the PITPα PtdIns/PtdCho exchange cycle and offer a rationale for the high conservation of particular sets of residues across evolutionarily distant members of the metazoan StART-like PITP family.
Assuntos
Bicamadas Lipídicas/metabolismo , Modelos Moleculares , Fosfatidilcolinas/metabolismo , Fosfatidilinositóis/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Apoproteínas/química , Apoproteínas/genética , Apoproteínas/metabolismo , Transporte Biológico , Biologia Computacional , Sequência Conservada , Transferência de Energia , Ligação de Hidrogênio , Ligantes , Bicamadas Lipídicas/química , Simulação de Dinâmica Molecular , Mutação de Sentido Incorreto , Fosfatidilcolinas/química , Fosfatidilinositóis/química , Proteínas de Transferência de Fosfolipídeos/química , Proteínas de Transferência de Fosfolipídeos/genética , Polimorfismo de Nucleotídeo Único , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
The Golgi complex constitutes a central way station of the eukaryotic endomembrane system, an intricate network of organelles engaged in control of membrane trafficking and the processing of various cellular components. Previous ideas of compartmental stability within this network are gradually being reshaped by concepts describing a biochemical continuum of hybrid organelles whose constitution is regulated by compartmental maturation. Membrane lipid composition and lipid signaling processes make fundamental contributions to compartmentalization strategies that are themselves critical for organizing cellular architecture and biochemical activities. Phosphatidylinositol transfer proteins (PITPs) are increasingly recognized as key regulators of membrane trafficking through the secretory pathway. They do so by coordinating lipid metabolism with lipid signaling, translating this information to core protein components of the membrane trafficking machinery. In this capacity, PITPs can be viewed as regulators of an essential lipid-protein interface of cisternal maturation. It is also now becoming appreciated, for the first time, that such an interface plays important roles in larger systems processes that link secretory pathway function with cell proliferation.
Assuntos
Complexo de Golgi/química , Complexo de Golgi/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Animais , Humanos , Proteínas de Transferência de Fosfolipídeos/químicaRESUMO
Synaptic adhesion-like molecules (SALMs) are a family of cell adhesion molecules involved in neurite outgrowth and synapse formation. Of the five family members, only SALM1, -2, and -3 contain a cytoplasmic C-terminal PDZ-binding motif. We have found that SALM1 is unique among the SALMs because deletion of its PDZ-binding motif (SALM1ΔPDZ) blocks its surface expression in heterologous cells. When expressed in hippocampal neurons, SALM1ΔPDZ had decreased surface expression in dendrites and the cell soma but not in axons, suggesting that the PDZ-binding domain may influence cellular trafficking of SALMs to specific neuronal locations. Endoglycosidase H digestion assays indicated that SALM1ΔPDZ is retained in the endoplasmic reticulum (ER) in heterologous cells. However, when the entire C-terminal tail of SALM1 was deleted, SALM1 was detected on the cell surface. Using serial deletions, we identified a region of SALM1 that contains a putative dileucine ER retention motif, which is not present in the other SALMs. Mutation of this DXXXLL motif allowed SALM1 to leave the ER and enhanced its surface expression in heterologous cells and neurons. An increase in the number of protrusions at the dendrites and cell body was observed when this SALM1 mutant was expressed in hippocampal neurons. With electron microscopy, these protrusions appeared to be irregular, enlarged spines and filopodia. Thus, enrichment of SALM1 on the cell surface affects dendritic arborization, and intracellular motifs regulate its dendritic versus axonal localization.
Assuntos
Axônios/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Dendritos/metabolismo , Hipocampo/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Axônios/ultraestrutura , Moléculas de Adesão Celular Neuronais/genética , Dendritos/ultraestrutura , Células HeLa , Hipocampo/citologia , Humanos , Proteínas do Tecido Nervoso/genética , Domínios PDZ , Transporte Proteico/fisiologia , Deleção de SequênciaRESUMO
Sorting nexin 27 (SNX27) is a 62-kDa protein localized to early endosomes and known to regulate the intracellular trafficking of ion channels and receptors. In addition to a PX domain, SNX27 is the only sorting family member that contains a PDZ domain. To identify novel SNX27-PDZ binding partners, we performed a proteomic screen in mouse principal kidney cortical collecting duct cells using a GST-SNX27 fusion construct as bait. We found that ß-Pix (p21-activated kinase-interactive exchange factor), a guanine nucleotide exchange factor for the Rho family of small GTPases known to regulate cell motility directly interacted with SNX27. The association of ß-Pix and SNX27 is specific for ß-Pix isoforms terminating in the type-1 PDZ binding motif (ETNL). In the same screen we also identified Git1/2 as a potential SNX27 interacting protein. The interaction between SNX27 and Git1/2 is indirect and mediated by ß-Pix. Furthermore, we show recruitment of the ß-Pix·Git complex to endosomal sites in a SNX27-dependent manner. Finally, migration assays revealed that depletion of SNX27 from HeLa and mouse principal kidney cortical collecting duct cells significantly decreases cell motility. We propose a model by which SNX27 regulates trafficking of ß-Pix to focal adhesions and thereby influences cell motility.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Túbulos Renais Coletores/metabolismo , Fosfoproteínas/metabolismo , Nexinas de Classificação/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Motivos de Aminoácidos , Animais , Proteínas de Ciclo Celular/genética , Movimento Celular/fisiologia , Adesões Focais/genética , Adesões Focais/metabolismo , Proteínas Ativadoras de GTPase/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Túbulos Renais Coletores/citologia , Camundongos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Células NIH 3T3 , Domínios PDZ , Fosfoproteínas/genética , Transporte Proteico , Fatores de Troca de Nucleotídeo Guanina Rho , Nexinas de Classificação/genéticaRESUMO
The polyisoprenoid diphosphates farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) are intermediates in the synthesis of cholesterol and related sterols by the mevalonate pathway and precursors for the addition of isoprenyl anchors to many membrane proteins. We developed tandem mass spectrometry assays to evaluate polyisoprenoid diphosphate phosphatase activity of an unusual integral membrane lipid enzyme: type 1 polyisoprenoid diphosphate phosphatase encoded by the PPAPDC2 gene (PDP1/PPAPDC2). In vitro, recombinant PDP1/PPAPDC2 preferentially hydrolyzed polyisoprenoid diphosphates, including FPP and GGPP over a variety of glycerol- and sphingo-phospholipid substrates. Overexpression of mammalian PDP1/PPAPDC2 in budding yeast depletes cellular pools of FPP leading to growth defects and sterol auxotrophy. In mammalian cells, PDP1/PPAPDC2 localizes to the endoplasmic reticulum and nuclear envelope and, unlike the structurally related lipid phosphate phosphatases, is predicted to be oriented with key residues of its catalytic domain facing the cytoplasmic face of the membrane. Studies using synthetic isoprenols with chemical properties that facilitate detection by mass spectrometry identify a pathway for interconversion of isoprenols and isoprenoid diphosphates in intact mammalian cells and demonstrate a role for PDP1/PPAPDC2 in this process. Overexpression of PDP1/PPAPDC2 in mammalian cells substantially decreases protein isoprenylation and results in defects in cell growth and cytoskeletal organization that are associated with dysregulation of Rho family GTPases. Taken together, these results focus attention on integral membrane lipid phosphatases as regulators of isoprenoid phosphate metabolism and suggest that PDP1/PPAPDC2 is a functional isoprenoid diphosphate phosphatase.
Assuntos
Retículo Endoplasmático/enzimologia , Monoéster Fosfórico Hidrolases/metabolismo , Fosfatos de Poli-Isoprenil/metabolismo , Prenilação de Proteína/fisiologia , Sesquiterpenos/metabolismo , Animais , Retículo Endoplasmático/genética , Células HeLa , Humanos , Insetos , Lipídeos de Membrana/genética , Lipídeos de Membrana/metabolismo , Monoéster Fosfórico Hidrolases/genética , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
Lipid phosphate monoesters including phosphatidic acid, lysophosphatidic acid, sphingosine 1-phosphate and ceramide 1-phosphate are intermediates in phosho- and sphingo-lipid biosynthesis and also play important roles in intra- and extra-cellular signaling. Dephosphorylation of these lipids terminates their signaling actions and, in some cases, generates products with additional biological activities or metabolic fates. The key enzymes responsible for dephosphorylation of these lipid phosphate substrates are collectively termed lipid phosphate phosphatases (LPPs). They are integral membrane enzymes with a core domain of six transmembrane spanning alpha-helices linked by extramembrane loops. LPPs are oriented in the membrane with their N- and C-termini facing the cytoplasm. LPPs exhibit isoform and cell specific localization patterns being variably distributed between endomembrane compartments (primarily the endoplasmic reticulum and Golgi apparatus) and the plasma membrane. The active site of these enzymes is formed from residues within two of the extramembrane loops and faces the lumen of endomembrane compartments or, when localized to the plasma membrane, towards, the extracellular space. Biochemical, pharmacological, cell biological and genetic studies identify roles for LPPs in both intracellular lipid metabolism and the regulation of both intra- and extra-cellular signaling pathways that control cell growth, survival and migration. This article describes procedures for the expression of LPPs in insect and mammalian cells and their analysis by SDS-PAGE and Western blotting. The most straightforward way to determine LPP activity is to measure release of the substrate phosphate group. We described methods for the synthesis and purification of [(32)P]-labeled LPP substrates. We describe the use of both radiolabeled and fluorescent lipid substrates for the detection, quantitation and analysis of the enzymatic activities of the LPPs measured using intact or broken cell preparations as the source of enzyme.
Assuntos
Bioquímica/métodos , Enzimas/metabolismo , Fosfatidato Fosfatase/análise , Animais , Cromatografia Líquida de Alta Pressão/instrumentação , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia em Camada Fina/instrumentação , Cromatografia em Camada Fina/métodos , Enzimas/genética , Corantes Fluorescentes , Insetos , Mamíferos , Micelas , Octoxinol/química , Fosfatidato Fosfatase/genética , Fosfatidato Fosfatase/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Phospholipids and sphingolipids play critical roles in signal transduction, intracellular membrane trafficking, and control of cell growth and survival. We discuss recent progress in the identification and characterization of a family of integral membrane proteins with central roles in bioactive lipid metabolism and signalling. These five groups of homologous proteins, which we collectively term LPTs (lipid phosphatases/phosphotransferases), are characterized by a core domain containing six transmembrane-spanning alpha-helices connected by extramembrane loops, two of which interact to form the catalytic site. LPT family members are localized to all major membrane compartments of the cell. The transmembrane topology of these proteins places their active site facing the lumen of endomembrane compartments or the extracellular face of the plasma membrane. Sequence conservation between the active site of the LPPs (lipid phosphate phosphatases), SPPs (sphingosine phosphate phosphatases) and the recently identified SMSs (sphingomyelin synthases) with vanadium-dependent fungal oxidases provides a framework for understanding their common catalytic mechanism. LPPs hydrolyse LPA (lysophosphatidic acid), S1P (sphingosine 1-phosphate) and structurally-related substrates. Although LPPs can dephosphorylate intracellularly generated substrates to control intracellular lipid metabolism and signalling, their best understood function is to regulate cell surface receptor-mediated signalling by LPA and S1P by inactivating these lipids at the plasma membrane or in the extracellular space. SPPs are intracellularly localized S1P-selective phosphatases, with key roles in the pathways of sphingolipid metabolism linked to control of cell growth and survival. The SMS enzymes catalyse the interconversion of phosphatidylcholine and ceramide with sphingomyelin and diacylglycerol, suggesting a pivotal role in both housekeeping lipid synthesis and regulation of bioactive lipid mediators. The remaining members of the LPT family, the LPR/PRGs (lipid phosphatase-related proteins/plasticity-related genes) and CSS2s (type 2 candidate sphingomyelin synthases), are presently much less well studied. These two groups include proteins that lack critical amino acids within the catalytic site, and could therefore not use the conserved LPT reaction mechanism to catalyse lipid phosphatase or phosphotransferase reactions. In this review, we discuss recent ideas about their possible biological activities and functions, which appear to involve regulation of cellular morphology and, possibly, lipid metabolism and signalling in the nuclear envelope.