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1.
J Biol Chem ; 284(37): 24881-90, 2009 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-19592499

RESUMO

Elevated sphingolipids have been associated with increased cardiovascular disease. Conversely, atherosclerosis is reduced in mice by blocking de novo synthesis of sphingolipids catalyzed by serine palmitoyltransferase (SPT). The SPT enzyme is composed of the SPTLC1 and -2 subunits, and here we describe a novel protein-protein interaction between SPTLC1 and the PDZ protein Par3 (partitioning defective protein 3). Mammalian SPTLC1 orthologs have a highly conserved C terminus that conforms to a type II PDZ protein interaction motif, and by screening PDZ domain protein arrays with an SPTLC1 C-terminal peptide, we found it bound the third PDZ domain of Par3. Overlay and immunoprecipitation assays confirmed this interaction and indicate Par3 is able to associate with the SPTLC1/2 holoenzyme by binding the C-terminal SPTLC1 PDZ motif. The physiologic existence of the SPTLC1/2-Par3 complex was detected in mouse liver and macrophages, and short interfering RNA inhibition of Par3 in human THP-1 monocytes significantly reduced SPT activity and de novo ceramide synthesis by nearly 40%. Given monocyte recruitment into inflamed vessels is thought to promote atherosclerosis, and because Par3 and sphingolipids have been associated with polarized cell migration, we tested whether the ability of THP-1 monocytes to migrate toward MCP-1 (monocyte chemoattractant protein 1) depended upon Par3 and SPTLC1 expression. Knockdown of Par3 significantly reduced MCP1-induced chemotaxis of THP-1 monocytes, as did knockdown of SPTLC1, and this Par3 effect depended upon SPT activity and was blunted by ceramide treatment. In conclusion, protein arrays were used to identify a novel SPTLC1-Par3 interaction that associates with increased monocyte serine palmitoyltransferase activity and chemotaxis toward inflammatory signals.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Quimiotaxia , Proteínas de Membrana/metabolismo , Monócitos/citologia , Serina C-Palmitoiltransferase/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Motivos de Aminoácidos , Sequência de Aminoácidos , Linhagem Celular , Movimento Celular , Humanos , Inflamação , Dados de Sequência Molecular , Monócitos/metabolismo , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Esfingolipídeos/metabolismo
2.
J Biol Chem ; 283(52): 36624-35, 2008 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-18957418

RESUMO

Harlequin ichthyosis is a congenital scaling syndrome of the skin in which affected infants have epidermal hyperkeratosis and a defective permeability barrier. Mutations in the gene encoding a member of the ABCA transporter family, ABCA12, have been linked to harlequin ichthyosis, but the molecular function of the protein is unknown. To investigate the activity of ABCA12, we generated Abca12 null mice and analyzed the impact on skin function and lipid content. Abca12-/- mice are born with a thickened epidermis and die shortly after birth, as water rapidly evaporates from their skin. In vivo skin proliferation measurements suggest a lack of desquamation of the skin cells, rather than enhanced proliferation of basal layer keratinocytes, accounts for the 5-fold thickening of the Abca12-/- stratum corneum. Electron microscopy revealed a loss of the lamellar permeability barrier in Abca12-/- skin. This was associated with a profound reduction in skin linoleic esters of long-chain omega-hydroxyceramides and a corresponding increase in their glucosyl ceramide precursors. Because omega-hydroxyceramides are required for the barrier function of the skin, these results establish that ABCA12 activity is required for the generation of long-chain ceramide esters that are essential for the development of normal skin structure and function.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Ceramidas/química , Ésteres/química , Lipídeos/química , Animais , Proliferação de Células , Ceramidas/metabolismo , Genótipo , Glucose/química , Heterozigoto , Ácido Linoleico/metabolismo , Camundongos , Microscopia Eletrônica , Modelos Genéticos , Permeabilidade , Pele/metabolismo
3.
Biochemistry ; 47(23): 6138-47, 2008 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-18484747

RESUMO

ABCA1 transport of cholesterol and phospholipids to nascent HDL particles plays a central role in lipoprotein metabolism and macrophage cholesterol homeostasis. ABCA1 activity is regulated both at the transcriptional level and at the post-translational level. To explore mechanisms involved in the post-translational regulation of the transporter, we have used affinity purification and mass spectrometry to identify proteins that bind ABCA1 and influence its activity. Previously, we demonstrated that an interaction between beta1-syntrophin stimulated ABCA1 activity, at least in part, be slowing the degradation of the transporter. This work demonstrates that one subunit of the serine palmitoyltransferase enzyme, SPTLC1, but not subunit 2 (SPTLC2), is copurified with ABCA1 and negatively regulates its function. In human THP-I macrophages and in mouse liver, the ABCA1-SPTLC1 complex was detected by co-immunoprecipitation, demonstrating that the interaction occurs in cellular settings where ABCA1 activity is critical for HDL genesis. Pharmacologic inhibition of SPTLC1 with myriocin, which resulted in the disruption of the SPTLC1-ABCA1 complex, and siRNA knockdown of SPTLC1 expression both stimulated ABCA1 efflux by nearly 60% ( p < 0.05). In contrast, dominant-negative mutants of SPTLC1 inhibited ABCA1 efflux, indicating that a reduced level of sphingomyelin synthesis could not explain the effect of myriocin on ABCA1 activity. In 293 cells, the SPTLC1 inhibition of ABCA1 activity led to the blockade of the exit of ABCA1 from the endoplasmic reticulum. In contrast, myriocin treatment of macrophages increased the level of cell surface ABCA1. In composite, these results indicate that the physical interaction of ABCA1 and SPTLC1 results in reduction of ABCA1 activity and that inhibition of this interaction produces enhanced cholesterol efflux.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Colesterol/metabolismo , Macrófagos/enzimologia , Serina C-Palmitoiltransferase/metabolismo , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Apolipoproteína A-I/metabolismo , Ácidos Graxos Monoinsaturados/farmacologia , Humanos , Cinética , Camundongos , Ligação Proteica , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Serina C-Palmitoiltransferase/antagonistas & inibidores
4.
J Lipid Res ; 48(3): 621-32, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17142808

RESUMO

The highly branched mammalian lung relies on surfactant, a mixture of phospholipids, cholesterol, and hydrophobic proteins, to reduce intraalveolar surface tension and prevent lung collapse. Human mutations in the ABCA3 transporter have been associated with childhood respiratory disease of variable severity and onset. Here, we report the generation of Abca3 null mice, which became lethargic and cyanotic and died within 1 h of birth. Tissue blots found ABCA3 expression was highest in lung but was also detectable in other tissues, including the kidney. Gross development of kidney and lung was normal in neonatal Abca3(-/-) pups, but the mice failed to inflate their lungs, leading to death from atelectatic respiratory failure. Ultrastructural analysis of the Abca3(-/-) lungs revealed an absence of surfactant from the alveolar space and a profound loss of mature lamellar bodies, the intracellular storage organelle for surfactant. Mass spectrometry measurement of >300 phospholipids in lung tissue taken from Abca3(-/-) mice showed a dramatic reduction of phosphatidylglycerol (PG) levels as well as selective reductions in phosphatidylcholine species containing short acyl chains. These results establish a requirement of ABCA3 for lamellar body formation and pulmonary surfactant secretion and suggest a unique and critical role for the transporter in the metabolism of pulmonary PG. They also demonstrate the utility of the Abca3 null mouse as a model for a devastating human disease.


Assuntos
Transportadores de Cassetes de Ligação de ATP/fisiologia , Fosfatidilgliceróis/metabolismo , Surfactantes Pulmonares/metabolismo , Insuficiência Respiratória/fisiopatologia , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Animais Recém-Nascidos , Colesterol/metabolismo , Homozigoto , Pulmão/embriologia , Pulmão/crescimento & desenvolvimento , Pulmão/ultraestrutura , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Modelos Genéticos , Mutação , Fosfolipídeos/metabolismo , Insuficiência Respiratória/genética , Insuficiência Respiratória/metabolismo
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