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1.
Nat Commun ; 12(1): 4718, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34354069

RESUMO

Phospholipid synthesis and fat storage as triglycerides are regulated by lipin phosphatidic acid phosphatases (PAPs), whose enzymatic PAP function requires association with cellular membranes. Using hydrogen deuterium exchange mass spectrometry, we find mouse lipin 1 binds membranes through an N-terminal amphipathic helix, the Ig-like domain and HAD phosphatase catalytic core, and a middle lipin (M-Lip) domain that is conserved in mammalian and mammalian-like lipins. Crystal structures of the M-Lip domain reveal a previously unrecognized protein fold that dimerizes. The isolated M-Lip domain binds membranes both in vitro and in cells through conserved basic and hydrophobic residues. Deletion of the M-Lip domain in lipin 1 reduces PAP activity, membrane association, and oligomerization, alters subcellular localization, diminishes acceleration of adipocyte differentiation, but does not affect transcriptional co-activation. This establishes the M-Lip domain as a dimeric protein fold that binds membranes and is critical for full functionality of mammalian lipins.


Assuntos
Fosfatidato Fosfatase/química , Células 3T3-L1 , Adipogenia , Sequência de Aminoácidos , Animais , Membrana Celular/metabolismo , Sequência Conservada , Cristalografia por Raios X , Células HEK293 , Humanos , Espectrometria de Massa com Troca Hidrogênio-Deutério , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Modelos Moleculares , Simulação de Dinâmica Molecular , Fosfatidato Fosfatase/genética , Fosfatidato Fosfatase/metabolismo , Ligação Proteica , Domínios Proteicos , Dobramento de Proteína , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Homologia de Sequência de Aminoácidos , Transcrição Genética
2.
Atherosclerosis ; 330: 76-84, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34256308

RESUMO

BACKGROUND AND AIMS: Atherosclerosis is the most prominent underlying cause of cardiovascular disease (CVD). It is initiated by cholesterol deposition in the arterial intima, which causes macrophage recruitment and proinflammatory responses that promote plaque growth, necrotic core formation, and plaque rupture. Lipin-1 is a phosphatidic acid phosphohydrolase for glycerolipid synthesis. We have shown that lipin-1 phosphatase activity promotes macrophage pro-inflammatory responses when stimulated with modified low-density lipoprotein (modLDL) and accelerates atherosclerosis. Lipin-1 also independently acts as a transcriptional co-regulator where it enhances the expression of genes involved in ß-oxidation. In hepatocytes and adipocytes, lipin-1 augments the activity of transcription factors such as peroxisome proliferator-activated receptor (PPARs). PPARs control the expression of anti-inflammatory genes in macrophages and slow or reduce atherosclerotic progression. Therefore, we hypothesize myeloid-derived lipin-1 transcriptional co-regulatory activity reduces atherosclerosis. METHODS: We used myeloid-derived lipin-1 knockout (lipin-1mKO) and littermate control mice and AAV8-PCSK9 along with high-fat diet to elicit atherosclerosis. RESULTS: Lipin-1mKO mice had larger aortic root plaques than littermate control mice after 8 and 12 weeks of a high-fat diet. Lipin-1mKO mice also had increased serum proinflammatory cytokine concentrations, reduced apoptosis in plaques, and larger necrotic cores in the plaques compared to control mice. CONCLUSIONS: Combined, the data suggest lipin-1 transcriptional co-regulatory activity in myeloid cells is atheroprotective.


Assuntos
Proteínas Nucleares , Fosfatidato Fosfatase/metabolismo , Pró-Proteína Convertase 9 , Animais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Nucleares/genética , Compostos Orgânicos , Fosfatidato Fosfatase/genética
3.
Nat Commun ; 12(1): 2813, 2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-34001876

RESUMO

Apicomplexa are obligate intracellular parasites responsible for major human diseases. Their intracellular survival relies on intense lipid synthesis, which fuels membrane biogenesis. Parasite lipids are generated as an essential combination of fatty acids scavenged from the host and de novo synthesized within the parasite apicoplast. The molecular and metabolic mechanisms allowing regulation and channeling of these fatty acid fluxes for intracellular parasite survival are currently unknown. Here, we identify an essential phosphatidic acid phosphatase in Toxoplasma gondii, TgLIPIN, as the central metabolic nexus responsible for controlled lipid synthesis sustaining parasite development. Lipidomics reveal that TgLIPIN controls the synthesis of diacylglycerol and levels of phosphatidic acid that regulates the fine balance of lipids between storage and membrane biogenesis. Using fluxomic approaches, we uncover the first parasite host-scavenged lipidome and show that TgLIPIN prevents parasite death by 'lipotoxicity' through effective channeling of host-scavenged fatty acids to storage triacylglycerols and membrane phospholipids.


Assuntos
Membrana Celular/metabolismo , Lipidômica/métodos , Fosfatidato Fosfatase/metabolismo , Fosfolipídeos/metabolismo , Proteínas de Protozoários/metabolismo , Toxoplasma/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Retículo Endoplasmático/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/parasitologia , Prepúcio do Pênis/citologia , Técnicas de Silenciamento de Genes , Homeostase/genética , Interações Hospedeiro-Parasita , Humanos , Masculino , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Fosfatidato Fosfatase/genética , Proteínas de Protozoários/genética , Toxoplasma/genética , Toxoplasma/ultraestrutura
4.
Nat Commun ; 12(1): 690, 2021 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-33514739

RESUMO

Lamins and transmembrane proteins within the nuclear envelope regulate nuclear structure and chromatin organization. Nuclear envelope transmembrane protein 39 (Net39) is a muscle nuclear envelope protein whose functions in vivo have not been explored. We show that mice lacking Net39 succumb to severe myopathy and juvenile lethality, with concomitant disruption in nuclear integrity, chromatin accessibility, gene expression, and metabolism. These abnormalities resemble those of Emery-Dreifuss muscular dystrophy (EDMD), caused by mutations in A-type lamins (LMNA) and other genes, like Emerin (EMD). We observe that Net39 is downregulated in EDMD patients, implicating Net39 in the pathogenesis of this disorder. Our findings highlight the role of Net39 at the nuclear envelope in maintaining muscle chromatin organization, gene expression and function, and its potential contribution to the molecular etiology of EDMD.


Assuntos
Proteínas de Membrana/deficiência , Músculo Esquelético/patologia , Distrofia Muscular de Emery-Dreifuss/genética , Membrana Nuclear/patologia , Proteínas Nucleares/metabolismo , Fosfatidato Fosfatase/metabolismo , Animais , Linhagem Celular , Cromatina/metabolismo , Sequenciamento de Cromatina por Imunoprecipitação , Modelos Animais de Doenças , Regulação para Baixo , Feminino , Humanos , Lamina Tipo A/genética , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Músculo Esquelético/citologia , Distrofia Muscular de Emery-Dreifuss/patologia , Proteínas Nucleares/genética , Fosfatidato Fosfatase/genética , RNA-Seq , Estudos Retrospectivos
5.
Cancer Sci ; 112(2): 792-802, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33274548

RESUMO

Phosphatidylinositol-3 kinase (PI3K) inhibitor and histone deacetylase (HDAC) inhibitor have been developed as potential anticancer drugs. However, the cytotoxicity of PI3K inhibitor or HDAC inhibitor alone is relatively weak. We recently developed a novel HDAC/PI3K dual inhibitor FK-A11 and confirmed its enhanced cytotoxicity when compared to that of PI3K inhibitor or HDAC inhibitor alone on several cancer cell lines. However, the in vivo antitumor activity of FK-A11 was insufficient. We conducted high-throughput RNA interfering screening and identified gene LPIN1 which enhances the cytotoxicity of FK-A11. Downregulation of LPIN1 enhanced simultaneous inhibition of HDAC and PI3K by FK-A11 and enhanced the cytotoxicity of FK-A11. Propranolol, a beta-adrenoreceptor which is also a LPIN1 inhibitor, enhanced the in vitro and in vivo cytotoxicity and antitumor effect of FK-A11. These findings should help in the development of FK-A11 as a novel HDAC/PI3K dual inhibitor.


Assuntos
Antineoplásicos/farmacologia , Inibidores de Histona Desacetilases/farmacologia , Fosfatidato Fosfatase/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Ensaios Antitumorais Modelo de Xenoenxerto
6.
BMC Genomics ; 21(1): 881, 2020 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-33297965

RESUMO

BACKGROUND: Bisphenol S (BPS) is a common bisphenol A (BPA) substitute, since BPA is virtually banned worldwide. However, BPS and BPA have both endocrine disrupting properties. Their effects appear mostly in adulthood following perinatal exposures. The objective of the present study was to investigate the impact of perinatal and chronic exposure to BPS at the low dose of 1.5 µg/kg body weight/day on the transcriptome and methylome of the liver in 23 weeks-old C57BL6/J male mice. RESULTS: This multi-omic study highlights a major impact of BPS on gene expression (374 significant deregulated genes) and Gene Set Enrichment Analysis show an enrichment focused on several biological pathways related to metabolic liver regulation. BPS exposure also induces a hypomethylation in 58.5% of the differentially methylated regions (DMR). Systematic connections were not found between gene expression and methylation profile excepted for 18 genes, including 4 genes involved in lipid metabolism pathways (Fasn, Hmgcr, Elovl6, Lpin1), which were downregulated and featured differentially methylated CpGs in their exons or introns. CONCLUSIONS: This descriptive study shows an impact of BPS on biological pathways mainly related to an integrative disruption of metabolism (energy metabolism, detoxification, protein and steroid metabolism) and, like most high-throughput studies, contributes to the identification of potential exposure biomarkers.


Assuntos
Metilação de DNA , Transcriptoma , Animais , Compostos Benzidrílicos , Feminino , Fígado/metabolismo , Masculino , Camundongos , Fenóis , Fosfatidato Fosfatase/metabolismo , Gravidez , Sulfonas
7.
Nat Commun ; 11(1): 5842, 2020 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-33203880

RESUMO

Increased lipogenesis has been linked to an increased cancer risk and poor prognosis; however, the underlying mechanisms remain obscure. Here we show that phosphatidic acid phosphatase (PAP) lipin-1, which generates diglyceride precursors necessary for the synthesis of glycerolipids, interacts with and is a direct substrate of the Src proto-oncogenic tyrosine kinase. Obesity-associated microenvironmental factors and other Src-activating growth factors, including the epidermal growth factor, activate Src and promote Src-mediated lipin-1 phosphorylation on Tyr398, Tyr413 and Tyr795 residues. The tyrosine phosphorylation of lipin-1 markedly increases its PAP activity, accelerating the synthesis of glycerophospholipids and triglyceride. Alteration of the three tyrosine residues to phenylalanine (3YF-lipin-1) disables lipin-1 from mediating Src-enhanced glycerolipid synthesis, cell proliferation and xenograft growth. Re-expression of 3YF-lipin-1 in PyVT;Lpin1-/- mice fails to promote progression and metastasis of mammary tumours. Human breast tumours exhibit increased p-Tyr-lipin-1 levels compared to the adjacent tissues. Importantly, statistical analyses show that levels of p-Tyr-lipin-1 correlate with tumour sizes, lymph node metastasis, time to recurrence and survival of the patients. These results illustrate a direct lipogenesis-promoting role of the pro-oncogenic Src, providing a mechanistic link between obesity-associated mitogenic signaling and breast cancer malignancy.


Assuntos
Neoplasias da Mama/patologia , Proteína Tirosina Quinase CSK/genética , Fosfatidato Fosfatase/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/mortalidade , Proteína Tirosina Quinase CSK/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Humanos , Lipogênese/fisiologia , Masculino , Neoplasias Mamárias Animais/genética , Neoplasias Mamárias Animais/patologia , Camundongos Mutantes , Camundongos Transgênicos , Fosfatidato Fosfatase/genética , Fosforilação , Tirosina/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Biomolecules ; 10(9)2020 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-32887262

RESUMO

Lipid phosphate phosphatases (LPPs) are a group of three enzymes (LPP1-3) that belong to a phospholipid phosphatase (PLPP) family. The LPPs dephosphorylate a wide spectrum of bioactive lipid phosphates, among which lysophosphatidate (LPA) and sphingosine 1-phosphate (S1P) are two important extracellular signaling molecules. The LPPs are integral membrane proteins, which are localized on plasma membranes and intracellular membranes, including the endoplasmic reticulum and Golgi network. LPPs regulate signaling transduction in cancer cells and demonstrate different effects in cancer progression through the breakdown of extracellular LPA and S1P and other intracellular substrates. This review is intended to summarize an up-to-date understanding about the functions of LPPs in cancers.


Assuntos
Neoplasias/enzimologia , Neoplasias/genética , Fosfatidato Fosfatase/genética , Fosfatidato Fosfatase/metabolismo , Animais , Membrana Celular/química , Membrana Celular/metabolismo , Humanos , Lisofosfolipídeos/genética , Lisofosfolipídeos/metabolismo , Fosfatidato Fosfatase/química , Transdução de Sinais , Esfingosina/análogos & derivados , Esfingosina/genética , Esfingosina/metabolismo , Regulação para Cima
9.
Sci Rep ; 10(1): 13209, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32764655

RESUMO

Ubiquitous PAP2 lipid phosphatases are involved in a wide array of central physiological functions. PgpB from Escherichia coli constitutes the archetype of this subfamily of membrane proteins. It displays a dual function by catalyzing the biosynthesis of two essential lipids, the phosphatidylglycerol (PG) and the undecaprenyl phosphate (C55-P). C55-P constitutes a lipid carrier allowing the translocation of peptidoglycan subunits across the plasma membrane. PG and C55-P are synthesized in a redundant manner by PgpB and other PAP2 and/or unrelated membrane phosphatases. Here, we show that PgpB is the sole, among these multiple phosphatases, displaying this dual activity. The inactivation of PgpB does not confer any apparent growth defect, but its inactivation together with another PAP2 alters the cell envelope integrity increasing the susceptibility to small hydrophobic compounds. Evidence is also provided of an interplay between PAP2s and the peptidoglycan polymerase PBP1A. In contrast to PGP hydrolysis, which relies on a His/Asp/His catalytic triad of PgpB, the mechanism of C55-PP hydrolysis appeared as only requiring the His/Asp diad, which led us to hypothesize distinct processes. Moreover, thermal stability analyses highlighted a substantial structural change upon phosphate binding by PgpB, supporting an induced-fit model of action.


Assuntos
Membrana Celular/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Redes e Vias Metabólicas , Fosfatidato Fosfatase/metabolismo , Motivos de Aminoácidos , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Técnicas de Inativação de Genes , Teste de Complementação Genética , Hidrólise , Proteínas de Membrana/metabolismo , Modelos Moleculares , Proteínas de Ligação às Penicilinas/metabolismo , Peptidoglicano Glicosiltransferase/metabolismo , Fosfatidato Fosfatase/genética , Fosfatidilgliceróis/metabolismo , Fosfatos de Poli-Isoprenil/metabolismo , Especificidade por Substrato , Termotolerância
10.
Biochem J ; 477(14): 2735-2754, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32648926

RESUMO

The mitogen-activated protein kinase (MAPK)-interacting kinases (MNKs) are serine/threonine protein kinases that are activated by the ERK1/2 (extracellular regulated kinase) and p38α/ß MAPK pathways. The MNKs have previously been implicated in metabolic disease and shown to mediate diet-induced obesity. In particular, knockout of MNK2 in mice protects from the weight gain induced by a high-fat diet. These and other data suggest that MNK2 regulates the expansion of adipose tissue (AT), a stable, long-term energy reserve that plays an important role in regulating whole-body energy homeostasis. Using the well-established mouse 3T3-L1 in vitro model of adipogenesis, the role of the MNKs in adipocyte differentiation and lipid storage was investigated. Inhibition of MNK activity using specific inhibitors failed to impair adipogenesis or lipid accumulation, suggesting that MNK activity is not required for adipocyte differentiation and does not regulate lipid storage. However, small-interfering RNA (siRNA) knock-down of MNK2 did reduce lipid accumulation and regulated the levels of two major lipogenic transcriptional regulators, ChREBP (carbohydrate response element-binding protein) and LPIN1 (Lipin-1). These factors are responsible for controlling the expression of genes for proteins involved in de novo lipogenesis and triglyceride synthesis. The knock-down of MNK2 also increased the expression of hormone-sensitive lipase which catalyses the breakdown of triglyceride. These findings identify MNK2 as a regulator of adipocyte metabolism, independently of its catalytic activity, and reveal some of the mechanisms by which MNK2 drives AT expansion. The development of an MNK2-targeted therapy may, therefore, be a useful intervention for reducing weight caused by excessive nutrient intake.


Assuntos
Adipócitos/metabolismo , Adipogenia/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Células 3T3-L1 , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Diferenciação Celular , Regulação da Expressão Gênica , Camundongos , Camundongos Knockout , Fosfatidato Fosfatase/genética , Fosfatidato Fosfatase/metabolismo , Proteínas Serina-Treonina Quinases/genética , RNA Interferente Pequeno
11.
Proc Natl Acad Sci U S A ; 117(30): 17832-17841, 2020 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-32661178

RESUMO

Spermatogonial stem cells (SSCs) are essential for the generation of sperm and have potential therapeutic value for treating male infertility, which afflicts >100 million men world-wide. While much has been learned about rodent SSCs, human SSCs remain poorly understood. Here, we molecularly characterize human SSCs and define conditions favoring their culture. To achieve this, we first identified a cell-surface protein, PLPPR3, that allowed purification of human primitive undifferentiated spermatogonia (uSPG) highly enriched for SSCs. Comparative RNA-sequencing analysis of these enriched SSCs with differentiating SPG (KIT+ cells) revealed the full complement of genes that shift expression during this developmental transition, including genes encoding key components in the TGF-ß, GDNF, AKT, and JAK-STAT signaling pathways. We examined the effect of manipulating these signaling pathways on cultured human SPG using both conventional approaches and single-cell RNA-sequencing analysis. This revealed that GDNF and BMP8B broadly support human SPG culture, while activin A selectively supports more advanced human SPG. One condition-AKT pathway inhibition-had the unique ability to selectively support the culture of primitive human uSPG. This raises the possibility that supplementation with an AKT inhibitor could be used to culture human SSCs in vitro for therapeutic applications.


Assuntos
Transdução de Sinais , Espermatogônias/citologia , Espermatogônias/metabolismo , Transcriptoma , Biomarcadores , Separação Celular , Células Cultivadas , Biologia Computacional , Imunofluorescência , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Imunofenotipagem , Masculino , Fosfatidato Fosfatase/genética , Fosfatidato Fosfatase/metabolismo
12.
Brain ; 143(6): 1746-1765, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32516804

RESUMO

TOR1A/TorsinA mutations cause two incurable diseases: a recessive congenital syndrome that can be lethal, and a dominantly-inherited childhood-onset dystonia (DYT-TOR1A). TorsinA has been linked to phosphatidic acid lipid metabolism in Drosophila melanogaster. Here we evaluate the role of phosphatidic acid phosphatase (PAP) enzymes in TOR1A diseases using induced pluripotent stem cell-derived neurons from patients, and mouse models of recessive Tor1a disease. We find that Lipin PAP enzyme activity is abnormally elevated in human DYT-TOR1A dystonia patient cells and in the brains of four different Tor1a mouse models. Its severity also correlated with the dosage of Tor1a/TOR1A mutation. We assessed the role of excess Lipin activity in the neurological dysfunction of Tor1a disease mouse models by interbreeding these with Lpin1 knock-out mice. Genetic reduction of Lpin1 improved the survival of recessive Tor1a disease-model mice, alongside suppressing neurodegeneration, motor dysfunction, and nuclear membrane pathology. These data establish that TOR1A disease mutations cause abnormal phosphatidic acid metabolism, and suggest that approaches that suppress Lipin PAP enzyme activity could be therapeutically useful for TOR1A diseases.


Assuntos
Chaperonas Moleculares/metabolismo , Fosfatidato Fosfatase/metabolismo , Animais , Encéfalo/patologia , Modelos Animais de Doenças , Distonia/genética , Distonia/metabolismo , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Chaperonas Moleculares/genética , Mutação , Neurônios/metabolismo , Fosfatidato Fosfatase/genética , Fosfatidato Fosfatase/fisiologia
13.
J Biol Chem ; 295(33): 11473-11485, 2020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32527729

RESUMO

The Nem1-Spo7 complex in the yeast Saccharomyces cerevisiae is a protein phosphatase that catalyzes the dephosphory-lation of Pah1 phosphatidate phosphatase, required for its translocation to the nuclear/endoplasmic reticulum membrane. The Nem1-Spo7/Pah1 phosphatase cascade plays a major role in triacylglycerol synthesis and in the regulation of phospholipid synthesis. In this work, we examined Spo7, a regulatory subunit required for Nem1 catalytic function, to identify residues that govern formation of the Nem1-Spo7 complex. By deletion analysis of Spo7, we identified a hydrophobic Leu-Leu-Ile (LLI) sequence comprising residues 54-56 as being required for the protein to complement the temperature-sensitive phenotype of an spo7Δ mutant strain. Mutational analysis of the LLI sequence with alanine and arginine substitutions showed that its overall hydrophobicity is crucial for the formation of the Nem1-Spo7 complex as well as for the Nem1 catalytic function on its substrate, Pah1, in vivo Consistent with the role of the Nem1-Spo7 complex in activating the function of Pah1, we found that the mutational effects of the Spo7 LLI sequence were on the Nem1-Spo7/Pah1 axis that controls lipid synthesis and related cellular processes (e.g. triacylglycerol/phospholipid synthesis, lipid droplet formation, nuclear/endoplasmic reticulum membrane morphology, vacuole fusion, and growth on glycerol medium). These findings advance the understanding of Nem1-Spo7 complex formation and its role in the phosphatase cascade that regulates the function of Pah1 phosphatidate phosphatase.


Assuntos
Proteínas de Membrana/metabolismo , Proteínas Nucleares/metabolismo , Fosfatidato Fosfatase/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Gotículas Lipídicas/metabolismo , Metabolismo dos Lipídeos , Proteínas de Membrana/química , Mapas de Interação de Proteínas , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/química
14.
Artigo em Inglês | MEDLINE | ID: mdl-32376340

RESUMO

The bioactive lipid lysophosphatidic acid (LPA) is emerging as an important mediator of inflammation in cardiovascular diseases. Produced in large part by the secreted lysophospholipase D autotaxin (ATX), LPA acts on a series of G protein-coupled receptors and may have action on atypical receptors such as RAGE to exert potent effects on vascular cells, including the promotion of foam cell formation and phenotypic modulation of smooth muscle cells. The signaling effects of LPA can be terminated by integral membrane lipid phosphate phosphatases (LPP) that hydrolyze the lipid to receptor inactive products. Human genetic variants in PLPP3, that predict lower levels of LPP3, associate with risk for premature coronary artery disease, and reductions of LPP3 expression in mice promote the development of experimental atherosclerosis and enhance inflammation in the atherosclerotic lesions. Recent evidence also supports a role for ATX, and potentially LPP3, in calcific aortic stenosis. In summary, LPA may be a relevant inflammatory mediator in atherosclerotic cardiovascular disease and heightened LPA signaling may explain the cardiovascular disease risk effect of PLPP3 variants.


Assuntos
Doenças Cardiovasculares/metabolismo , Inflamação/metabolismo , Lisofosfolipídeos/metabolismo , Músculo Liso Vascular/metabolismo , Fosfatidato Fosfatase/metabolismo , Transdução de Sinais , Humanos , Lisofosfolipídeos/química , Estrutura Molecular , Fosfatidato Fosfatase/genética
15.
Essays Biochem ; 64(3): 547-563, 2020 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-32451553

RESUMO

Extracellular lysophosphatidate (LPA) signalling is regulated by the balance of LPA formation by autotaxin (ATX) versus LPA degradation by lipid phosphate phosphatases (LPP) and by the relative expressions of six G-protein-coupled LPA receptors. These receptors increase cell proliferation, migration, survival and angiogenesis. Acute inflammation produced by tissue damage stimulates ATX production and LPA signalling as a component of wound healing. If inflammation does not resolve, LPA signalling becomes maladaptive in conditions including arthritis, neurologic pain, obesity and cancers. Furthermore, LPA signalling through LPA1 receptors promotes fibrosis in skin, liver, kidneys and lungs. LPA also promotes the spread of tumours to other organs (metastasis) and the pro-survival properties of LPA explain why LPA counteracts the effects of chemotherapeutic agents and radiotherapy. ATX is secreted in response to radiation-induced DNA damage during cancer treatments and this together with increased LPA1 receptor expression leads to radiation-induced fibrosis. The anti-inflammatory agent, dexamethasone, decreases levels of inflammatory cytokines/chemokines. This is linked to a coordinated decrease in the production of ATX and LPA1/2 receptors and increased LPA degradation through LPP1. These effects explain why dexamethasone attenuates radiation-induced fibrosis. Increased LPA signalling is also associated with cardiovascular disease including atherosclerosis and deranged LPA signalling is associated with pregnancy complications including preeclampsia and intrahepatic cholestasis of pregnancy. LPA contributes to chronic inflammation because it stimulates the secretion of inflammatory cytokines/chemokines, which increase further ATX production and LPA signalling. Attenuating maladaptive LPA signalling provides a novel means of treating inflammatory diseases that underlie so many important medical conditions.


Assuntos
Doenças Cardiovasculares/metabolismo , Fibrose Pulmonar Idiopática/metabolismo , Lisofosfolipídeos/metabolismo , Neoplasias/metabolismo , Complicações na Gravidez/metabolismo , Transdução de Sinais , Cicatrização/fisiologia , Animais , Feminino , Humanos , Inflamação/metabolismo , Fosfatidato Fosfatase/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Gravidez , Receptores Acoplados a Proteínas G/metabolismo
16.
Front Immunol ; 11: 787, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32431707

RESUMO

Macrophage responses contribute to a diverse array of pathologies ranging from infectious disease to sterile inflammation. Polarization of macrophages determines their cellular function within biological processes. Lipin-1 is a phosphatidic acid phosphatase in which its enzymatic activity contributes to macrophage pro-inflammatory responses. Lipin-1 also possesses transcriptional co-regulator activity and whether this activity is required for macrophage polarization is unknown. Using mice that lack only lipin-1 enzymatic activity or both enzymatic and transcriptional coregulator activities from myeloid cells, we investigated the contribution of lipin-1 transcriptional co-regulator function toward macrophage wound healing polarization. Macrophages lacking both lipin-1 activities did not elicit IL-4 mediated gene expression to levels seen in either wild-type or lipin-1 enzymatically deficient macrophages. Furthermore, mice lacking myeloid-associated lipin-1 have impaired full thickness excisional wound healing compared to wild-type mice or mice only lacking lipin-1 enzymatic activity from myeloid cell. Our study provides evidence that lipin-1 transcriptional co-regulatory activity contributes to macrophage polarization and influences wound healing in vivo.


Assuntos
Polaridade Celular/genética , Interleucina-4/metabolismo , Ativação de Macrófagos , Macrófagos/imunologia , Fosfatidato Fosfatase/metabolismo , Animais , Polaridade Celular/imunologia , Células Cultivadas , Expressão Gênica , Técnicas de Inativação de Genes , Inflamação/genética , Inflamação/imunologia , Interleucina-4/genética , Macrófagos/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfatidato Fosfatase/genética , Cicatrização/genética , Cicatrização/imunologia
17.
Mol Cell Endocrinol ; 508: 110793, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32229289

RESUMO

AIM: Cardiovascular complication is a major cause of mortality and morbidity in patients with diabetes. Insulin sensitivity loss is a major contributor to the pathogenesis of cardiovascular diseases in diabetes. Based on our previous research, diacylglycerol (DAG) levels play an important role in high saturated fatty acid-induced insulin resistance. Phosphatidic acid phosphatase (LPP3), a key enzyme for synthesizing DAG, is indispensable for normal cardiac functions and vascular health. However, adipose knockdown of LPP3 increases insulin sensitivity, suggesting that LPP3 regulation may be complicated in hearts. The aim of this study was to investigate LPP3 roles in diabetic cardiac insulin sensitivity and to identify potential upstream targets implicated in diabetic cardiomyopathy. METHODS AND RESULTS: Mice were fed a high fat diet (HF) or a low fat diet (control) for up to 24 weeks. After 24 weeks, we found that high fat diet-induced cardiac dysfunction is linked to elevated LPP3 compared to the control group (P < 0.05). In addition, knockdown of LPP3 rescued the glucose uptake that was impaired by palmitate treatment alone in cardiomyoblasts (P < 0.05). Furthermore, we identified miR-184 as an upstream regulator targeting LPP3 and further confirmed the link between DAG and insulin sensitivity. MiR-184 mimic transfection rescued the glucose uptake and glucose consumption that had been impaired by palmitate treatment alone (P < 0.05). CONCLUSION: In hearts of high fat diet-fed mice, increased LPP3 contributes to insulin resistance via increased DAG levels. A small non-coding RNA, miR-184, at least partially regulates this signal pathway to alleviate insulin resistance.


Assuntos
Dieta Hiperlipídica , Diglicerídeos/metabolismo , Coração/fisiopatologia , Resistência à Insulina/genética , MicroRNAs/metabolismo , Miócitos Cardíacos/metabolismo , Fosfatidato Fosfatase/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Masculino , Camundongos Endogâmicos C57BL , MicroRNAs/genética
18.
Mol Med Rep ; 22(1): 559-565, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32319636

RESUMO

Hypoxia-inducible factor (HIF)-1α is a transcription factor that is activated in low oxygen conditions. Adipose tissues are poorly oxygenated in patients with obesity. The low oxygen conditions in obese adipose tissues induce HIF­1α in adipocytes. Previous studies using genetically modified mice suggest that HIF­1α contributes to dysfunction in adipocytes. Lipin1 is a bifunctional protein that works as a phosphatidate phosphatase and transcriptional coactivator, which regulates lipid metabolism and adipogenesis, respectively. HIF­1α directly regulates Lipin1 in hepatocytes. However, the regulation of Lipin1 by HIF­1α in adipocytes is not well determined. Therefore, the present study investigated the regulation of Lipin1 by HIF­1α in adipocytes. Expression levels of Lipin1 were reduced in epididymal adipose tissues of adipocyte­specific HIF­1α knockout mice, indicating that HIF­1α regulates Lipin1 in adipocytes. In differentiated mature adipocytes, a HIF­1α activator, dimethyloxallyl glycine (DMOG), was demonstrated to increase Lipin1, and a HIF­1α inhibitor, 3­(5'­hydroxymethyl­2'­furyl)-1­benzylindazole (YC­1), reversed this increase, indicating that HIF­1α regulates Lipin1 in differentiated adipocytes. However, during differentiation of pre­adipocytes into adipocytes, YC­1 increased Lipin1 even though HIF­1α was decreased. The differentiation efficiency increased with YC­1 treatment. In addition, DMOG reduced Lipin1 expression levels during differentiation despite increased HIF­1α. Under these conditions, differentiation efficiency was reduced. These results suggest that Lipin1 is negatively regulated by HIF­1α in pre­adipocytes. Our results show that regulation of Lipin1 by HIF­1α is different in adipocytes and pre­adipocytes.


Assuntos
Adipócitos/citologia , Adipogenia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Fosfatidato Fosfatase/metabolismo , Células 3T3-L1 , Adipócitos/metabolismo , Animais , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Camundongos , Camundongos Knockout
19.
FEBS J ; 287(22): 4848-4861, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32150788

RESUMO

Germline mutation in the PTEN gene is the genetic basis of PTEN hamartoma tumor syndrome with the affected individuals harboring features of autism spectrum disorders. Characterizing a panel of 14 autism-associated PTEN missense mutations revealed reduced protein stability, catalytic activity, and subcellular distribution. Nine out of 14 (64%) PTEN missense mutants had reduced protein expression with most mutations confined to the C2 domain. Selected mutants displayed enhanced polyubiquitination and shortened protein half-life, but that did not appear to involve the polyubiquitination sites at lysine residues at codon 13 or 289. Analyzing their intrinsic lipid phosphatase activities revealed that 78% (11 out of 14) of these mutants had twofold to 10-fold reduction in catalytic activity toward phosphatidylinositol phosphate substrates. Analyzing the subcellular localization of the PTEN missense mutants showed that 64% (nine out of 14) had altered nuclear-to-cytosol ratios with four mutants (G44D, H123Q, E157G, and D326N) showing greater nuclear localization. The E157G mutant was knocked-in to an induced pluripotent stem cell line and recapitulated a similar nuclear targeting preference. Furthermore, iPSCs expressing the E157G mutant were more proliferative at the neural progenitor cell stage but exhibited more extensive dendritic outgrowth. In summary, the combination of biological changes in PTEN is expected to contribute to the behavioral and cellular features of this neurodevelopmental disorder.


Assuntos
Transtorno do Espectro Autista/genética , Núcleo Celular/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Mutação de Sentido Incorreto , Crescimento Neuronal/genética , PTEN Fosfo-Hidrolase/genética , Transtorno do Espectro Autista/metabolismo , Western Blotting , Linhagem Celular Tumoral , Células HEK293 , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células PC-3 , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidato Fosfatase/genética , Fosfatidato Fosfatase/metabolismo , Fosforilação , Estabilidade Proteica
20.
Nat Commun ; 11(1): 1309, 2020 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-32161260

RESUMO

Lipin/Pah phosphatidic acid phosphatases (PAPs) generate diacylglycerol to regulate triglyceride synthesis and cellular signaling. Inactivating mutations cause rhabdomyolysis, autoinflammatory disease, and aberrant fat storage. Disease-mutations cluster within the conserved N-Lip and C-Lip regions that are separated by 500-residues in humans. To understand how the N-Lip and C-Lip combine for PAP function, we determined crystal structures of Tetrahymena thermophila Pah2 (Tt Pah2) that directly fuses the N-Lip and C-Lip. Tt Pah2 adopts a two-domain architecture where the N-Lip combines with part of the C-Lip to form an immunoglobulin-like domain and the remaining C-Lip forms a HAD-like catalytic domain. An N-Lip C-Lip fusion of mouse lipin-2 is catalytically active, which suggests mammalian lipins function with the same domain architecture as Tt Pah2. HDX-MS identifies an N-terminal amphipathic helix essential for membrane association. Disease-mutations disrupt catalysis or destabilize the protein fold. This illustrates mechanisms for lipin/Pah PAP function, membrane association, and lipin-related pathologies.


Assuntos
Fosfatidato Fosfatase/metabolismo , Fosfatidato Fosfatase/ultraestrutura , Proteínas de Protozoários/ultraestrutura , Domínio Catalítico/genética , Cristalografia por Raios X , Células HEK293 , Humanos , Fosfatidato Fosfatase/genética , Fosfatidato Fosfatase/isolamento & purificação , Conformação Proteica em alfa-Hélice , Proteínas de Protozoários/genética , Proteínas de Protozoários/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/ultraestrutura , Tetrahymena thermophila/enzimologia , Transfecção
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