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1.
Infect Immun ; 89(2)2021 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-33199355

RESUMO

Cholera toxin (CT) is an AB5 protein toxin that activates the stimulatory alpha subunit of the heterotrimeric G protein (Gsα) through ADP-ribosylation. Activation of Gsα produces a cytopathic effect by stimulating adenylate cyclase and the production of cAMP. To reach its cytosolic Gsα target, CT binds to the plasma membrane of a host cell and travels by vesicle carriers to the endoplasmic reticulum (ER). The catalytic CTA1 subunit then exploits the quality control mechanism of ER-associated degradation to move from the ER to the cytosol. ER-associated degradation is functionally linked to another quality control system, the unfolded protein response (UPR). However, the role of the UPR in cholera intoxication is unclear. We report here that CT triggers the UPR after 4 h of toxin exposure. A functional toxin was required to induce the UPR, but, surprisingly, activation of the adenylate cyclase signaling pathway was not sufficient to trigger the process. Toxin-induced activation of the UPR coincided with increased toxin accumulation in the cytosol. Chemical activation of the heterotrimeric G protein or the UPR also enhanced the onset of CTA1 delivery to the cytosol, thus producing a toxin-sensitive phenotype. These results indicate there is a cAMP-independent response to CT that activates the UPR and thereby enhances the efficiency of intoxication.


Assuntos
Fator 6 Ativador da Transcrição/metabolismo , Toxina da Cólera/metabolismo , Toxina da Cólera/toxicidade , Imunidade/efeitos dos fármacos , Resposta a Proteínas não Dobradas/fisiologia , Vibrio cholerae/imunologia , Vibrio cholerae/patogenicidade
2.
J Lipid Res ; 60(2): 242-268, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30504233

RESUMO

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/química
3.
J Biol Chem ; 292(35): 14438-14455, 2017 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-28718450

RESUMO

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/metabolismo
4.
J Lipid Res ; 57(8): 1492-506, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27256690

RESUMO

A reliable method for purifying envelope-stripped nuclei from immortalized murine embryonic fibroblasts (iMEFs) was established. Quantitative profiling of the glycerophospholipids (GPLs) in envelope-free iMEF nuclei yields several conclusions. First, we find the endonuclear glycerophospholipidome differs from that of bulk membranes, and phosphatidylcholine (PtdCho) and phosphatidylethanolamine species are the most abundant endonuclear GPLs by mass. By contrast, phosphatidylinositol (PtdIns) represents a minor species. We also find only a slight enrichment of saturated versus unsaturated GPL species in iMEF endonuclear fractions. Moreover, much lower values for GPL mass were measured in the iMEF nuclear matrix than those reported for envelope-stripped IMF-32 nuclei. The collective results indicate that the nuclear matrix in these cells is a GPL-poor environment where GPL occupies only approximately 0.1% of the total nuclear matrix volume. This value suggests GPL accommodation in this compartment can be satisfied by binding to resident proteins. Finally, we find no significant role for the PtdIns/PtdCho-transfer protein, PITPα, in shuttling PtdIns into the iMEF nuclear matrix.


Assuntos
Fibroblastos/metabolismo , Membrana Nuclear/metabolismo , Fosfolipídeos/metabolismo , Animais , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Células Cultivadas , Embrião de Mamíferos/química , Fibroblastos/ultraestrutura , Camundongos , Proteínas de Transferência de Fosfolipídeos/metabolismo
5.
Biochim Biophys Acta ; 1851(6): 724-35, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25592381

RESUMO

Phosphatidylinositol is a metabolic precursor of phosphoinositides and soluble inositol phosphates. Both sets of molecules represent versatile intracellular chemical signals in eukaryotes. While much effort has been invested in understanding the enzymes that produce and consume these molecules, central aspects for how phosphoinositide production is controlled and functionally partitioned remain unresolved and largely unappreciated. It is in this regard that phosphatidylinositol (PtdIns) transfer proteins (PITPs) are emerging as central regulators of the functional channeling of phosphoinositide pools produced on demand for specific signaling purposes. The physiological significance of these proteins is amply demonstrated by the consequences that accompany deficits in individual PITPs. Although the biological problem is fascinating, and of direct relevance to disease, PITPs remain largely uncharacterized. Herein, we discuss our perspectives regarding what is known about how PITPs work as molecules, and highlight progress in our understanding of how PITPs are integrated into cellular physiology. This article is part of a Special Issue entitled Phosphoinositides.


Assuntos
Fosfatidilinositóis/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Biológico , CDP-Diacilglicerol-Inositol 3-Fosfatidiltransferase/genética , CDP-Diacilglicerol-Inositol 3-Fosfatidiltransferase/metabolismo , Regulação da Expressão Gênica , Humanos , Metabolismo dos Lipídeos , Modelos Moleculares , Proteínas de Transferência de Fosfolipídeos/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Saccharomyces cerevisiae/genética , Transdução de Sinais , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/metabolismo
6.
bioRxiv ; 2023 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-36712082

RESUMO

Phosphoinositide metabolism defines the foundation of a major signaling pathway that is conserved throughout the eukaryotic kingdom. The 4-OH phosphorylated phosphoinositides such as phosphatidylinositol-4-phosphate (PtdIns4P) and phosphatidylinositol-4,5-bisphosphate are particularly important molecules as these execute intrinsically essential activities required for the viability of all eukaryotic cells studied thus far. Using intracellular tachyzoites of the apicomplexan parasite Toxoplasma gondii as model for assessing primordial roles for PtdIns4P signaling, we demonstrate the presence of PtdIns4P pools in Golgi/trans-Golgi (TGN) system and in post-TGN compartments of the parasite. Moreover, we show that deficits in PtdIns4P signaling result in structural perturbation of compartments that house dense granule cargo with accompanying deficits in dense granule exocytosis. Taken together, the data report a direct role for PtdIns4P in dense granule biogenesis and exocytosis. The data further indicate that the biogenic pathway for secretion-competent dense granule formation in T. gondii is more complex than simple budding of fully matured dense granules from the TGN.

7.
Front Cell Dev Biol ; 11: 1163574, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37791074

RESUMO

Phosphoinositides are a biologically essential class of phospholipids that contribute to organelle membrane identity, modulate membrane trafficking pathways, and are central components of major signal transduction pathways that operate on the cytosolic face of intracellular membranes in eukaryotes. Apicomplexans (such as Toxoplasma gondii and Plasmodium spp.) are obligate intracellular parasites that are important causative agents of disease in animals and humans. Recent advances in molecular and cell biology of Apicomplexan parasites reveal important roles for phosphoinositide signaling in key aspects of parasitosis. These include invasion of host cells, intracellular survival and replication, egress from host cells, and extracellular motility. As Apicomplexans have adapted to the organization of essential signaling pathways to accommodate their complex parasitic lifestyle, these organisms offer experimentally tractable systems for studying the evolution, conservation, and repurposing of phosphoinositide signaling. In this review, we describe the regulatory mechanisms that control the spatial and temporal regulation of phosphoinositides in the Apicomplexan parasites Plasmodium and T. gondii. We further discuss the similarities and differences presented by Apicomplexan phosphoinositide signaling relative to how these pathways are regulated in other eukaryotic organisms.

8.
J Cell Biol ; 219(5)2020 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-32303746

RESUMO

The yeast phosphatidylserine (PtdSer) decarboxylase Psd2 is proposed to engage in a membrane contact site (MCS) for PtdSer decarboxylation to phosphatidylethanolamine (PtdEtn). This proposed MCS harbors Psd2, the Sec14-like phosphatidylinositol transfer protein (PITP) Sfh4, the Stt4 phosphatidylinositol (PtdIns) 4-OH kinase, the Scs2 tether, and an uncharacterized protein. We report that, of these components, only Sfh4 and Stt4 regulate Psd2 activity in vivo. They do so via distinct mechanisms. Sfh4 operates via a mechanism for which its PtdIns-transfer activity is dispensable but requires an Sfh4-Psd2 physical interaction. The other requires Stt4-mediated production of PtdIns-4-phosphate (PtdIns4P), where Stt4 (along with the Sac1 PtdIns4P phosphatase and endoplasmic reticulum-plasma membrane tethers) indirectly modulate Psd2 activity via a PtdIns4P homeostatic mechanism that influences PtdSer accessibility to Psd2. These results identify an example in which the biological function of a Sec14-like PITP is cleanly uncoupled from its canonical in vitro PtdIns-transfer activity and challenge popular functional assumptions regarding lipid-transfer protein involvements in MCS function.


Assuntos
Proteínas de Membrana/genética , Fosfatidilserinas/genética , Proteínas de Transferência de Fosfolipídeos/genética , Proteínas de Saccharomyces cerevisiae/genética , 1-Fosfatidilinositol 4-Quinase/genética , Transporte Biológico/genética , Metabolismo dos Lipídeos/genética , Fosfatidiletanolaminas/genética , Fosfatidiletanolaminas/metabolismo , Fosfatidilserinas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
9.
Dev Cell ; 21(5): 810-2, 2011 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-22075144

RESUMO

Kim et al. (2011) challenge the dogma that phosphatidylinositol synthesis is restricted to the endoplasmic reticulum (ER) by showing that a mobile membrane compartment transports phosphatidylinositol synthase from the ER to numerous cellular compartments, including the plasma membrane. These findings significantly impact our view of phosphoinositide signaling in the cell.

10.
Clin Lipidol ; 5(6): 867-897, 2010 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-21603057

RESUMO

Inositol and phosphoinositide signaling pathways represent major regulatory systems in eukaryotes. The physiological importance of these pathways is amply demonstrated by the variety of diseases that involve derangements in individual steps in inositide and phosphoinositide production and degradation. These diseases include numerous cancers, lipodystrophies and neurological syndromes. Phosphatidylinositol transfer proteins (PITPs) are emerging as fascinating regulators of phosphoinositide metabolism. Recent advances identify PITPs (and PITP-like proteins) to be coincidence detectors, which spatially and temporally coordinate the activities of diverse aspects of the cellular lipid metabolome with phosphoinositide signaling. These insights are providing new ideas regarding mechanisms of inherited mammalian diseases associated with derangements in the activities of PITPs and PITP-like proteins.

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