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1.
Proc Natl Acad Sci U S A ; 116(16): 8038-8047, 2019 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-30944221

RESUMO

Abdominal aortic aneurysm (AAA) is an inflammatory vascular disease with high mortality and limited treatment options. How blood lipids regulate AAA development is unknown. Here lipidomics and genetic models demonstrate a central role for procoagulant enzymatically oxidized phospholipids (eoxPL) in regulating AAA. Specifically, through activating coagulation, eoxPL either promoted or inhibited AAA depending on tissue localization. Ang II administration to ApoE-/- mice increased intravascular coagulation during AAA development. Lipidomics revealed large numbers of eoxPL formed within mouse and human AAA lesions. Deletion of eoxPL-generating enzymes (Alox12 or Alox15) or administration of the factor Xa inhibitor rivaroxaban significantly reduced AAA. Alox-deficient mice displayed constitutively dysregulated hemostasis, including a consumptive coagulopathy, characterized by compensatory increase in prothrombotic aminophospholipids (aPL) in circulating cell membranes. Intravenously administered procoagulant PL caused clotting factor activation and depletion, induced a bleeding defect, and significantly reduced AAA development. These data suggest that Alox deletion reduces AAA through diverting coagulation away from the vessel wall due to eoxPL deficiency, instead activating clotting factor consumption and depletion in the circulation. In mouse whole blood, ∼44 eoxPL molecular species formed within minutes of clot initiation. These were significantly elevated with ApoE-/- deletion, and many were absent in Alox-/- mice, identifying specific eoxPL that modulate AAA. Correlation networks demonstrated eoxPL belonged to subfamilies defined by oxylipin composition. Thus, procoagulant PL regulate AAA development through complex interactions with clotting factors. Modulation of the delicate balance between bleeding and thrombosis within either the vessel wall or circulation was revealed that can either drive or prevent disease development.


Assuntos
Aorta Abdominal/fisiopatologia , Aneurisma da Aorta Abdominal , Fosfolipídeos , Angiotensinas/metabolismo , Animais , Aneurisma da Aorta Abdominal/genética , Aneurisma da Aorta Abdominal/metabolismo , Aneurisma da Aorta Abdominal/fisiopatologia , Fatores de Coagulação Sanguínea/genética , Fatores de Coagulação Sanguínea/metabolismo , Modelos Animais de Doenças , Feminino , Lipoxigenase/genética , Lipoxigenase/metabolismo , Masculino , Camundongos , Camundongos Knockout para ApoE , Fosfolipídeos/genética , Fosfolipídeos/metabolismo
2.
J Biol Chem ; 291(26): 13448-64, 2016 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-27129261

RESUMO

Eicosanoids are important mediators of fever, pain, and inflammation that modulate cell signaling during acute and chronic disease. We show by using lipidomics that thrombin-activated human platelets generate a new type of eicosanoid that both stimulates and primes human neutrophil integrin (Mac-1) expression, in response to formylmethionylleucylphenylalanine. Detailed characterization proposes a dioxolane structure, 8-hydroxy-9,11-dioxolane eicosatetraenoic acid (dioxolane A3, DXA3). The lipid is generated in nanogram amounts by platelets from endogenous arachidonate during physiological activation, with inhibition by aspirin in vitro or in vivo, implicating cyclooxygenase-1 (COX). Pharmacological and genetic studies on human/murine platelets revealed that DXA3 formation requires protease-activated receptors 1 and 4, cytosolic phospholipase A2 (cPLA2), Src tyrosine kinases, p38 MAPK, phospholipase C, and intracellular calcium. From data generated by purified COX isoforms and chemical oxidation, we propose that DXA3 is generated by release of an intermediate from the active site followed by oxygenation at C8. In summary, a new neutrophil-activating platelet-derived lipid generated by COX-1 is presented that can activate or prime human neutrophils, suggesting a role in innate immunity and acute inflammation.


Assuntos
Plaquetas/enzimologia , Ciclo-Oxigenase 1/metabolismo , Dioxolanos/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Ativação de Neutrófilo/fisiologia , Neutrófilos/metabolismo , Animais , Aspirina/farmacologia , Plaquetas/imunologia , Ciclo-Oxigenase 1/imunologia , Dioxolanos/imunologia , Imunidade Inata/efeitos dos fármacos , Imunidade Inata/fisiologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Antígeno de Macrófago 1/imunologia , Antígeno de Macrófago 1/metabolismo , Masculino , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Ativação de Neutrófilo/efeitos dos fármacos , Neutrófilos/imunologia , Ativação Plaquetária/efeitos dos fármacos , Ativação Plaquetária/fisiologia
3.
J Biol Chem ; 289(35): 24091-101, 2014 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-25008319

RESUMO

Remodeling of collagen by matrix metalloproteinases (MMPs) is crucial to tissue homeostasis and repair. MMP-13 is a collagenase with a substrate preference for collagen II over collagens I and III. It recognizes a specific, well-known site in the tropocollagen molecule where its binding locally perturbs the triple helix, allowing the catalytic domain of the active enzyme to cleave the collagen α chains sequentially, at Gly(775)-Leu(776) in collagen II. However, the specific residues upon which collagen recognition depends within and surrounding this locus have not been systematically mapped. Using our triple-helical peptide Collagen Toolkit libraries in solid-phase binding assays, we found that MMP-13 shows little affinity for Collagen Toolkit III, but binds selectively to two triple-helical peptides of Toolkit II. We have identified the residues required for the adhesion of both proMMP-13 and MMP-13 to one of these, Toolkit peptide II-44, which contains the canonical collagenase cleavage site. MMP-13 was unable to bind to a linear peptide of the same sequence as II-44. We also discovered a second binding site near the N terminus of collagen II (starting at helix residue 127) in Toolkit peptide II-8. The pattern of binding of the free hemopexin domain of MMP-13 was similar to that of the full-length enzyme, but the free catalytic subunit bound none of our peptides. The susceptibility of Toolkit peptides to proteolysis in solution was independent of the very specific recognition of immobilized peptides by MMP-13; the enzyme proved able to cleave a range of dissolved collagen peptides.


Assuntos
Colágeno Tipo II/metabolismo , Metaloproteinase 13 da Matriz/metabolismo , Peptídeos/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Colágeno Tipo II/química , Primers do DNA , Metaloproteinase 13 da Matriz/química , Dados de Sequência Molecular , Proteólise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Especificidade por Substrato
4.
J Lipid Res ; 54(11): 3085-97, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23883581

RESUMO

Oxidized phospholipids (oxPLs) generated nonenzymatically display pleiotropic biological actions in inflammation. Their generation by cellular cyclooxygenases (COXs) is currently unknown. To determine whether platelets generate prostaglandin (PG)-containing oxPLs, then characterize their structures and mechanisms of formation, we applied precursor scanning-tandem mass spectrometry to lipid extracts of agonist-activated human platelets. Thrombin, collagen, or ionophore activation stimulated generation of families of PGs comprising PGE2 and D2 attached to four phosphatidylethanolamine (PE) phospholipids (16:0p/, 18:1p/, 18:0p/, and 18:0a/). They formed within 2 to 5 min of activation in a calcium, phospholipase C, p38 MAP kinases, MEK1, cPLA2, and src tyrosine kinase-dependent manner (28.1 ± 2.3 pg/2 × 108 platelets). Unlike free PGs, they remained cell associated, suggesting an autocrine mode of action. Their generation was inhibited by in vivo aspirin supplementation (75 mg/day) or in vitro COX-1 blockade. Inhibitors of fatty acyl reesterification blocked generation significantly, while purified COX-1 was unable to directly oxidize PE in vitro. This indicates that they form in platelets via rapid esterification of COX-1 derived PGE2/D2 into PE. In summary, COX-1 in human platelets acutely mediates membrane phospholipid oxidation via formation of PG-esterified PLs in response to pathophysiological agonists.


Assuntos
Aspirina/farmacologia , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Ciclo-Oxigenase 1/metabolismo , Inibidores de Ciclo-Oxigenase/farmacologia , Fosfolipídeos/metabolismo , Prostaglandinas/metabolismo , Plaquetas/fisiologia , Cálcio/metabolismo , Dinoprostona/metabolismo , Relação Dose-Resposta a Droga , Esterificação/efeitos dos fármacos , Retroalimentação Fisiológica/efeitos dos fármacos , Humanos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , MAP Quinase Quinase 1/metabolismo , Fosfatidiletanolaminas/metabolismo , Ativação Plaquetária/efeitos dos fármacos , Prostaglandina D2/metabolismo , Proteína Quinase C/metabolismo , Receptor PAR-1/metabolismo , Trombina/metabolismo , Quinases da Família src/metabolismo
5.
Proc Natl Acad Sci U S A ; 106(11): 4195-200, 2009 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-19251642

RESUMO

Collagen and fibronectin are major components of vertebrate extracellular matrices. Their association and distribution control the development and properties of diverse tissues, but thus far no structural information has been available for the complex formed. Here, we report binding of a peptide, derived from the alpha(1) chain of type I collagen, to the gelatin-binding domain of human fibronectin and present the crystal structure of this peptide in complex with the (8-9)FnI domain pair. Both gelatin-binding domain subfragments, (6)FnI(1-2)FnII(7)FnI and (8-9)FnI, bind the same specific sequence on D-period 4 of collagen I alpha(1), adjacent to the MMP-1 cleavage site. (8-9)FnI also binds the equivalent sequence of the alpha(2) chain. The collagen peptide adopts an antiparallel beta-strand conformation, similar to structures of proteins from pathogenic bacteria bound to FnI domains. Analysis of the type I collagen sequence suggests multiple putative fibronectin-binding sites compatible with our structural model. We demonstrate, by kinetic unfolding experiments, that the triple-helical collagen state is destabilized by (8-9)FnI. This finding suggests a role for fibronectin in collagen proteolysis and tissue remodeling.


Assuntos
Colágeno Tipo I/química , Fibronectinas/química , Desnaturação Proteica , Sítios de Ligação , Colágeno Tipo I/metabolismo , Cristalografia por Raios X , Fibronectinas/metabolismo , Humanos , Fragmentos de Peptídeos , Ligação Proteica , Conformação Proteica
6.
PLoS One ; 15(10): e0240189, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33031441

RESUMO

Tissue factor (TF) is critical for the activation of blood coagulation. TF function is regulated by the amount of externalised phosphatidylserine (PS) and phosphatidylethanolamine (PE) on the surface of the cell in which it is expressed. We investigated the role PS and PE in fibroblast TF function. Fibroblasts expressed 6-9 x 104 TF molecules/cell but had low specific activity for FXa generation. We confirmed that this was associated with minimal externalized PS and PE and characterised for the first time the molecular species of PS/PE demonstrating that these differed from those found in platelets. Mechanical damage of fibroblasts, used to simulate vascular injury, increased externalized PS/PE and led to a 7-fold increase in FXa generation that was inhibited by annexin V and an anti-TF antibody. Platelet-derived extracellular vesicles (EVs), that did not express TF, supported minimal FVIIa-dependent FXa generation but substantially increased fibroblast TF activity. This enhancement in fibroblast TF activity could also be achieved using synthetic liposomes comprising 10% PS without TF. In conclusion, despite high levels of surface TF expression, healthy fibroblasts express low levels of external-facing PS and PE limiting their ability to generate FXa. Addition of platelet-derived TF-negative EVs or artificial liposomes enhanced fibroblast TF activity in a PS dependent manner. These findings contribute information about the mechanisms that control TF function in the fibroblast membrane.


Assuntos
Vesículas Extracelulares/metabolismo , Fibroblastos/metabolismo , Tromboplastina/metabolismo , Coagulação Sanguínea , Plaquetas/metabolismo , Linhagem Celular , Humanos , Lipossomos/metabolismo , Fosfatidiletanolaminas/metabolismo , Fosfatidilserinas/metabolismo , Tromboplastina/genética
7.
Circ Genom Precis Med ; 13(3): e002806, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32396387

RESUMO

BACKGROUND: Common chromosome 9p21 single nucleotide polymorphisms (SNPs) increase coronary heart disease risk, independent of traditional lipid risk factors. However, lipids comprise large numbers of structurally related molecules not measured in traditional risk measurements, and many have inflammatory bioactivities. Here, we applied lipidomic and genomic approaches to 3 model systems to characterize lipid metabolic changes in common Chr9p21 SNPs, which confer ≈30% elevated coronary heart disease risk associated with altered expression of ANRIL, a long ncRNA. METHODS: Untargeted and targeted lipidomics was applied to plasma from NPHSII (Northwick Park Heart Study II) homozygotes for AA or GG in rs10757274, followed by correlation and network analysis. To identify candidate genes, transcriptomic data from shRNA downregulation of ANRIL in HEK-293 cells was mined. Transcriptional data from vascular smooth muscle cells differentiated from induced pluripotent stem cells of individuals with/without Chr9p21 risk, nonrisk alleles, and corresponding knockout isogenic lines were next examined. Last, an in-silico analysis of miRNAs was conducted to identify how ANRIL might control lysoPL (lysophosphospholipid)/lysoPA (lysophosphatidic acid) genes. RESULTS: Elevated risk GG correlated with reduced lysoPLs, lysoPA, and ATX (autotaxin). Five other risk SNPs did not show this phenotype. LysoPL-lysoPA interconversion was uncoupled from ATX in GG plasma, suggesting metabolic dysregulation. Significantly altered expression of several lysoPL/lysoPA metabolizing enzymes was found in HEK cells lacking ANRIL. In the vascular smooth muscle cells data set, the presence of risk alleles associated with altered expression of several lysoPL/lysoPA enzymes. Deletion of the risk locus reversed the expression of several lysoPL/lysoPA genes to nonrisk haplotype levels. Genes that were altered across both cell data sets were DGKA, MBOAT2, PLPP1, and LPL. The in-silico analysis identified 4 ANRIL-regulated miRNAs that control lysoPL genes as miR-186-3p, miR-34a-3p, miR-122-5p, and miR-34a-5p. CONCLUSIONS: A Chr9p21 risk SNP associates with complex alterations in immune-bioactive phospholipids and their metabolism. Lipid metabolites and genomic pathways associated with coronary heart disease pathogenesis in Chr9p21 and ANRIL-associated disease are demonstrated.


Assuntos
Cromossomos Humanos Par 9/genética , Doença das Coronárias , Lisofosfolipídeos , Diester Fosfórico Hidrolases , Polimorfismo de Nucleotídeo Único , Cromossomos Humanos Par 9/metabolismo , Doença das Coronárias/genética , Doença das Coronárias/metabolismo , Células HEK293 , Humanos , Lisofosfolipídeos/genética , Lisofosfolipídeos/metabolismo , Masculino , Pessoa de Meia-Idade , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo
8.
Int J Biochem Cell Biol ; 40(10): 2253-63, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18436470

RESUMO

To assess the impact collagen structures may have on glycation, the effects of glucose upon bovine serum albumin, guinea pig skin collagen, rat tail tendon and monomeric collagen were compared under near physiological conditions. Proteins were incubated with or without 50 mM glucose for 64 d in pH 7.4 50 mM phosphate buffer, followed by reduction, acid/alkaline hydrolysis, and analysis. Yields of non-reducible fructose-lysine, in the form of the acid-degradation products furosine and pyridosine, were significantly higher from skin collagen when compared to albumin. Yields of reducible fructose-lysine, in the form of glucitol- and mannitol-lysine, were conversely much greater for albumin, while tail tendon reported intermediate values. Fructose-lysine and unmodified lysine within collagen fibres prior to incubation was therefore protected by the tight packing of the collagen helices, where milling of tail tendon to increase the surface area exposed much of it to reduction protocols. Together with an analysis of pentosidine formation and other products, these results have shown that the interior of the tightly packed skin collagen fibres is protected from both glycation and reduction, and that glycation products differ depending on the protein incubated. Amino acid analysis then showed that our glycated skin collagen was similar to human diabetic skin collagen. Significant quantities of glucose-independent unknowns form in control incubations; their composition again being protein-dependent. The four compound Ks as previously reported were found to be unique to glycated rat tail tendon and soluble collagen, while another glycation product detected in collagen but not albumin may be attributable to carboxymethyl-arginine.


Assuntos
Colágenos Fibrilares/metabolismo , Aminoácidos/análise , Animais , Arginina/análogos & derivados , Arginina/metabolismo , Boroidretos/farmacologia , Soluções Tampão , Carboidratos , Cromatografia Líquida de Alta Pressão , Colágenos Fibrilares/química , Colágenos Fibrilares/isolamento & purificação , Glucose/metabolismo , Glicosilação , Cobaias , Humanos , Hidrólise , Lisina/análogos & derivados , Lisina/metabolismo , Espectroscopia de Ressonância Magnética , Oxirredução/efeitos dos fármacos , Ratos , Pele/química
9.
Sci Rep ; 8(1): 13809, 2018 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-30218106

RESUMO

Fibrillar collagens have mechanical and biological roles, providing tissues with both tensile strength and cell binding sites which allow molecular interactions with cell-surface receptors such as integrins. A key question is: how do collagens allow tissue flexibility whilst maintaining well-defined ligand binding sites? Here we show that proline residues in collagen glycine-proline-hydroxyproline (Gly-Pro-Hyp) triplets provide local conformational flexibility, which in turn confers well-defined, low energy molecular compression-extension and bending, by employing two-dimensional 13C-13C correlation NMR spectroscopy on 13C-labelled intact ex vivo bone and in vitro osteoblast extracellular matrix. We also find that the positions of Gly-Pro-Hyp triplets are highly conserved between animal species, and are spatially clustered in the currently-accepted model of molecular ordering in collagen type I fibrils. We propose that the Gly-Pro-Hyp triplets in fibrillar collagens provide fibril "expansion joints" to maintain molecular ordering within the fibril, thereby preserving the structural integrity of ligand binding sites.


Assuntos
Colágeno/química , Colágeno/metabolismo , Prolina/metabolismo , Sequência de Aminoácidos , Aminoácidos/metabolismo , Animais , Feminino , Colágenos Fibrilares/metabolismo , Colágenos Fibrilares/fisiologia , Glicina/química , Hidroxiprolina/química , Espectroscopia de Ressonância Magnética , Camundongos , Camundongos Endogâmicos C57BL , Osteoblastos/metabolismo , Peptídeos/química , Prolina/fisiologia , Conformação Proteica , Ovinos
10.
JCI Insight ; 3(6)2018 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-29563336

RESUMO

Hemostatic defects are treated using coagulation factors; however, clot formation also requires a procoagulant phospholipid (PL) surface. Here, we show that innate immune cell-derived enzymatically oxidized phospholipids (eoxPL) termed hydroxyeicosatetraenoic acid-phospholipids (HETE-PLs) restore hemostasis in human and murine conditions of pathological bleeding. HETE-PLs abolished blood loss in murine hemophilia A and enhanced coagulation in factor VIII- (FVIII-), FIX-, and FX-deficient human plasma . HETE-PLs were decreased in platelets from patients after cardiopulmonary bypass (CPB). To explore molecular mechanisms, the ability of eoxPL to stimulate individual isolated coagulation factor/cofactor complexes was tested in vitro. Extrinsic tenase (FVIIa/tissue factor [TF]), intrinsic tenase (FVIIIa/FIXa), and prothrombinase (FVa/FXa) all were enhanced by both HETE-PEs and HETE-PCs, suggesting a common mechanism involving the fatty acid moiety. In plasma, 9-, 15-, and 12-HETE-PLs were more effective than 5-, 11-, or 8-HETE-PLs, indicating positional isomer specificity. Coagulation was enhanced at lower lipid/factor ratios, consistent with a more concentrated area for protein binding. Surface plasmon resonance confirmed binding of FII and FX to HETE-PEs. HETE-PEs increased membrane curvature and thickness, but not surface charge or homogeneity, possibly suggesting increased accessibility to cations/factors. In summary, innate immune-derived eoxPL enhance calcium-dependent coagulation factor function, and their potential utility in bleeding disorders is proposed.


Assuntos
Fatores de Coagulação Sanguínea/metabolismo , Hemorragia/enzimologia , Hemorragia/metabolismo , Fosfolipídeos/metabolismo , Trombina/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Coagulação Sanguínea , Fatores de Coagulação Sanguínea/genética , Plaquetas , Ponte Cardiopulmonar/efeitos adversos , Proteínas de Transporte , Cisteína Endopeptidases , Fator IX/genética , Fator VIII/genética , Fator VIIa/metabolismo , Fator X/genética , Hemofilia A , Hemorragia/prevenção & controle , Hemostasia , Humanos , Ácidos Hidroxieicosatetraenoicos , Lipoproteínas/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Proteínas de Neoplasias , Ressonância de Plasmônio de Superfície , Tromboplastina/antagonistas & inibidores , Tromboplastina/metabolismo
11.
J Mol Biol ; 359(2): 289-98, 2006 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-16631195

RESUMO

We report a rapid method to synthesize cystine cross-linked heterotrimeric collagenous peptides. They can be engineered to favour one particular axial alignment of the strands, called the register of the helix. Here, the sequence of the constituent peptides contains 18 residues of "guest" collagen type I sequence flanked by N and C-terminal (Gly-Pro-Pro)5 "host" modules which ensure helicity. Further C-terminal residues include appropriately spaced cysteine residues and alanine to provide the necessary flexibility for helix formation. The cross-linking reaction and subsequent separation protocols have been designed for any inserted collagen sequence that does not contain a cysteine residue. Mass spectrometry and ion-exchange chromatography allow us to distinguish between different disulphide-bonded species and to monitor the formation of side-products. Starting peptide can be recovered simply from the reaction mixture by reduction and separation. Yields are typically 30%, working on a 10 mg scale. 15N-1H NMR and platelet adhesion studies show that the peptide heterotrimers presented here can reshuffle to cover all three axial registers. Less flexible spacers between the disulphide linkages and the helix will restrict each heterotrimer to one register only.


Assuntos
Colágeno Tipo I/química , Colágeno Tipo I/síntese química , Integrina alfa2beta1/metabolismo , Peptídeos/química , Peptídeos/síntese química , Conformação Proteica , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cromatografia/métodos , Cromatografia Líquida de Alta Pressão , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Reagentes de Ligações Cruzadas/química , Cistina/química , Integrina alfa2beta1/química , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Peptídeos/metabolismo , Adesividade Plaquetária , Proteínas Recombinantes de Fusão/síntese química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência
12.
JCI Insight ; 2(7): e91634, 2017 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-28405621

RESUMO

Accurate and high-quality curation of lipidomic datasets generated from plasma, cells, or tissues is becoming essential for cell biology investigations and biomarker discovery for personalized medicine. However, a major challenge lies in removing artifacts otherwise mistakenly interpreted as real lipids from large mass spectrometry files (>60 K features), while retaining genuine ions in the dataset. This requires powerful informatics tools; however, available workflows have not been tailored specifically for lipidomics, particularly discovery research. We designed LipidFinder, an open-source Python workflow. An algorithm is included that optimizes analysis based on users' own data, and outputs are screened against online databases and categorized into LIPID MAPS classes. LipidFinder outperformed three widely used metabolomics packages using data from human platelets. We show a family of three 12-hydroxyeicosatetraenoic acid phosphoinositides (16:0/, 18:1/, 18:0/12-HETE-PI) generated by thrombin-activated platelets, indicating crosstalk between eicosanoid and phosphoinositide pathways in human cells. The software is available on GitHub (https://github.com/cjbrasher/LipidFinder), with full userguides.


Assuntos
Plaquetas/química , Eicosanoides/análise , Metabolômica/métodos , Fosfatidilinositóis/análise , Software , Humanos , Espectrometria de Massas , Fluxo de Trabalho
13.
Redox Biol ; 11: 663-672, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28160743

RESUMO

Activated platelets generate an eicosanoid proposed to be 8-hydroxy-9,10-dioxolane A3 (DXA3). Herein, we demonstrate that significant amounts of DXA3 are rapidly attached to phosphatidylethanolamine (PE) forming four esterified eicosanoids, 16:0p, 18:0p, 18:1p and 18:0a/DXA3-PEs that can activate neutrophil integrin expression. These lipids comprise the majority of DXA3 generated by platelets, are formed in ng amounts (24.3±6.1ng/2×108) and remain membrane bound. Pharmacological studies revealed DXA3-PE formation involves cyclooxygenase-1 (COX), protease-activated receptors (PAR) 1 and 4, cytosolic phospholipase A2 (cPLA2), phospholipase C and intracellular calcium. They are generated primarily via esterification of newly formed DXA3, but can also be formed in vitro via co-oxidation of PE during COX-1 co-oxidation of arachidonate. All four DXA3-PEs were detected in human clots. Purified platelet DXA3-PE activated neutrophil Mac-1 expression, independently of its hydrolysis to the free eicosanoid. This study demonstrates the structures and cellular synthetic pathway for a family of leukocyte-activating platelet phospholipids generated on acute activation, adding to the growing evidence that enzymatic PE oxidation is a physiological event in innate immune cells.


Assuntos
Plaquetas/metabolismo , Dioxolanos/sangue , Integrinas/sangue , Lipídeos/sangue , Fosfatidiletanolaminas/sangue , Cálcio/sangue , Ciclo-Oxigenase 1/sangue , Eicosanoides/sangue , Regulação da Expressão Gênica , Humanos , Integrinas/biossíntese , Antígeno de Macrófago 1/genética , Neutrófilos/metabolismo , Oxirredução , Fosfolipases A2 Citosólicas/sangue , Ativação Plaquetária/genética , Receptor PAR-1/sangue , Receptores de Trombina/sangue , Trombina/metabolismo , Fosfolipases Tipo C/sangue
14.
Sci Signal ; 10(507)2017 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-29184033

RESUMO

Blood coagulation functions as part of the innate immune system by preventing bacterial invasion, and it is critical to stopping blood loss (hemostasis). Coagulation involves the external membrane surface of activated platelets and leukocytes. Using lipidomic, genetic, biochemical, and mathematical modeling approaches, we found that enzymatically oxidized phospholipids (eoxPLs) generated by the activity of leukocyte or platelet lipoxygenases (LOXs) were required for normal hemostasis and promoted coagulation factor activities in a Ca2+- and phosphatidylserine (PS)-dependent manner. In wild-type mice, hydroxyeicosatetraenoic acid-phospholipids (HETE-PLs) enhanced coagulation and restored normal hemostasis in clotting-deficient animals genetically lacking p12-LOX or 12/15-LOX activity. Murine platelets generated 22 eoxPL species, all of which were missing in the absence of p12-LOX. Humans with the thrombotic disorder antiphospholipid syndrome (APS) had statistically significantly increased HETE-PLs in platelets and leukocytes, as well as greater HETE-PL immunoreactivity, than healthy controls. HETE-PLs enhanced membrane binding of the serum protein ß2GP1 (ß2-glycoprotein 1), an event considered central to the autoimmune reactivity responsible for APS symptoms. Correlation network analysis of 47 platelet eoxPL species in platelets from APS and control subjects identified their enzymatic origin and revealed a complex network of regulation, with the abundance of 31 p12-LOX-derived eoxPL molecules substantially increased in APS. In summary, circulating blood cells generate networks of eoxPL molecules, including HETE-PLs, which change membrane properties to enhance blood coagulation and contribute to the excessive clotting and immunoreactivity of patients with APS.


Assuntos
Fatores de Coagulação Sanguínea/metabolismo , Cálcio/metabolismo , Membrana Celular/metabolismo , Hemostasia , Fosfolipídeos/metabolismo , Ativação Plaquetária , Adulto , Idoso , Animais , Síndrome Antifosfolipídica/sangue , Síndrome Antifosfolipídica/enzimologia , Coagulação Sanguínea , Membrana Celular/ultraestrutura , Estudos de Coortes , Modelos Animais de Doenças , Feminino , Humanos , Ácidos Hidroxieicosatetraenoicos/análise , Ácidos Hidroxieicosatetraenoicos/metabolismo , Lipoxigenases/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Modelos Teóricos , Fosfolipídeos/análise , Trombose Venosa/sangue , Trombose Venosa/induzido quimicamente , Trombose Venosa/enzimologia , beta 2-Glicoproteína I/metabolismo
15.
Cell Metab ; 23(5): 930-44, 2016 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-27133131

RESUMO

Human platelets acutely increase mitochondrial energy generation following stimulation. Herein, a lipidomic circuit was uncovered whereby the substrates for this are exclusively provided by cPLA2, including multiple fatty acids and oxidized species that support energy generation via ß-oxidation. This indicates that acute lipid membrane remodeling is required to support energetic demands during platelet activation. Phospholipase activity is linked to energy metabolism, revealing cPLA2 as a central regulator of both lipidomics and energy flux. Using a lipidomic approach (LipidArrays), we also estimated the total number of lipids in resting, thrombin-activated, and aspirinized platelets. Significant diversity between genetically unrelated individuals and a wealth of species was revealed. Resting platelets demonstrated ∼5,600 unique species, with only ∼50% being putatively identified. Thrombin elevated ∼900 lipids >2-fold with 86% newly appearing and 45% inhibited by aspirin supplementation, indicating COX-1 is required for major activation-dependent lipidomic fluxes. Many lipids were structurally identified. With ∼50% of the lipids being absent from databases, a major opportunity for mining lipids relevant to human health and disease is presented.


Assuntos
Plaquetas/metabolismo , Metabolismo Energético , Metaboloma , Mitocôndrias/metabolismo , Fosfolipases A2 Citosólicas/metabolismo , Aspirina/farmacologia , Plaquetas/efeitos dos fármacos , Eicosanoides/metabolismo , Metabolismo Energético/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Ácidos Graxos/metabolismo , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Metaboloma/efeitos dos fármacos , Oxirredução , Fosfolipídeos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Especificidade por Substrato/efeitos dos fármacos , Trombina/farmacologia , Fatores de Tempo
16.
J Mol Biol ; 329(1): 175-83, 2003 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-12742026

RESUMO

Heating and subsequent cooling mixtures of (Pro-Pro-Gly)(10) and (Pro-Hyp-Gly)(10) peptides leads to formation of model heterotrimeric collagen helices that can be isolated by HPLC. These heterotrimeric collagen peptide helices are shown to be fundamentally unstable as denaturing then renaturing experiments result in heterotrimeric/homotrimeric mixtures. As the proportion of hydroxyproline-containing chains in the trimers increases, differential scanning calorimetry shows that the helix melting temperatures and denaturation enthalpies increasing non-linearly. Three types of Rich-Crick hydrogen bonds observed by NMR allow modelling of heterotrimeric structures based on published homotrimeric X-ray data. This revealed a small axial movement of (Pro-Hyp-Gly)(10) chains towards the C-terminal of the helix, demonstrating heterotrimeric asymmetry.


Assuntos
Colágeno/química , Oligopeptídeos/química , Peptídeos/química , Sequência de Aminoácidos , Varredura Diferencial de Calorimetria , Cromatografia Líquida de Alta Pressão , Colágeno/metabolismo , Ligação de Hidrogênio , Cinética , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Oligopeptídeos/metabolismo , Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica , Difração de Raios X
17.
Sci Rep ; 5: 12556, 2015 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-26220399

RESUMO

Collagens, the most abundant proteins in mammals, are defined by their triple-helical structures and distinctive Gly-Xaa-Yaa repeating sequence, where Xaa is often proline and Yaa, hydroxyproline (Hyp/O). It is known that hydroxyproline in the Yaa position stabilises the triple helix, and that lack of proline hydroxylation in vivo leads to dysfunctional collagen extracellular matrix assembly, due to a range of factors such as a change in hydration properties. In addition, we note that in model peptides, when Yaa is unmodified proline, the Xaa proline has a strong propensity to adopt an endo ring conformation, whilst when Yaa is hydroxyproline, the Xaa proline adopts a range of endo and exo conformations. Here we use a combination of solid-state NMR spectroscopy and potential energy landscape modelling of synthetic triple-helical collagen peptides to understand this effect. We show that hydroxylation of the Yaa proline causes the Xaa proline ring conformation to become metastable, which in turn confers flexibility on the triple helix.


Assuntos
Colágeno/química , Hidroxiprolina/química , Hidroxilação , Espectroscopia de Ressonância Magnética/métodos , Modelos Moleculares , Peptídeos/química , Prolina/química , Estrutura Secundária de Proteína
18.
Science ; 344(6185): 742-6, 2014 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-24833391

RESUMO

Nuclear magnetic resonance (NMR) spectroscopy is useful to determine molecular structure in tissues grown in vitro only if their fidelity, relative to native tissue, can be established. Here, we use multidimensional NMR spectra of animal and in vitro model tissues as fingerprints of their respective molecular structures, allowing us to compare the intact tissues at atomic length scales. To obtain spectra from animal tissues, we developed a heavy mouse enriched by about 20% in the NMR-active isotopes carbon-13 and nitrogen-15. The resulting spectra allowed us to refine an in vitro model of developing bone and to probe its detailed structure. The identification of an unexpected molecule, poly(adenosine diphosphate ribose), that may be implicated in calcification of the bone matrix, illustrates the analytical power of this approach.


Assuntos
Desenvolvimento Ósseo , Calcificação Fisiológica , Ressonância Magnética Nuclear Biomolecular/métodos , Poli Adenosina Difosfato Ribose/análise , Animais , Isótopos de Carbono , Matriz Extracelular/química , Lâmina de Crescimento/crescimento & desenvolvimento , Camundongos , Modelos Biológicos , Isótopos de Nitrogênio , Ovinos
19.
Peptides ; 36(1): 86-93, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22555281

RESUMO

Recently, the ability of polymeric collagen-like peptides to regulate cell behavior has generated great interest. A triple-helical peptide known as collagen-related peptide (CRP) contains the sequence (Gly-Pro-Hyp)(10). With Gly-Pro-Cys triplets appended to both of its termini, designated CRP(cys), chemical cross-linking using heterobifunctional reagents generates CRP(cys)-XL, a potent, widely used, polymeric agonist for platelet Glycoprotein VI, whereas non-cross-linked, monomeric CRP(cys) antagonizes Glycoprotein VI. Here, we describe how cysteine in these triplets may also undergo random air-induced oxidation, especially upon prolonged storage or repeated freeze-thawing, to form disulphide bonds, resulting in a lesser degree of polymerization than with chemical cross-linking. We investigated the monomeric and polymeric states of these and other cysteine-containing collagen-derived peptides, using gel filtration and dynamic light scattering, allowing the size of a CRP-XL aggregate to be estimated. The effect of cysteine thiols upon peptide adsorption to surfaces and subsequent platelet responses was investigated. This demonstrated that cysteine is required for strong binding to glass coverslips and to plastic plates used in ELISA assays.


Assuntos
Proteínas de Transporte/química , Cisteína/química , Peptídeos/química , Adsorção , Motivos de Aminoácidos , Sequência de Aminoácidos , Plaquetas/efeitos dos fármacos , Plaquetas/fisiologia , Proteínas de Transporte/isolamento & purificação , Proteínas de Transporte/farmacologia , Adesão Celular/efeitos dos fármacos , Cromatografia em Gel , Humanos , Proteínas Imobilizadas , Luz , Dados de Sequência Molecular , Oxirredução , Tamanho da Partícula , Peptídeos/isolamento & purificação , Peptídeos/farmacologia , Ligação Proteica , Estrutura Terciária de Proteína , Espalhamento de Radiação , Estreptavidina/química
20.
Biomaterials ; 32(27): 6621-32, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21663955

RESUMO

Collagen is the fundamental structural protein, comprising 25-35% of the total body protein, its rod-like triple helix providing support in many tissues. Our laboratory has synthesised 113 Toolkit peptides, each 63 residues long, covering the entirety of the homotrimeric helix sequence of collagen II and collagen III. These are used primarily to investigate protein-collagen interactions, from which biomedical applications are under development. Upon increasing the temperature of a Toolkit peptide solution, a novel low temperature transition (LTT) as well as a broadening of the helix unfolding higher temperature transition (HTT) was observed. Here, we hypothesized that unfolding of imperfect helices can account for the LTT. Peptides of various purities were isolated by HPLC or gel filtration, and their unfolding measured by polarimetry, CD, and DSC. The resulting temperature transitions were fitted to a kinetic unfolding equation, allowing comparison of the data, and explanation of the observed melting curve complexity as due to peptide imperfections. Finally, using a mathematical model, this data can be replicated by setting a parameter that quantifies the mutual stabilization conferred by helices on each side of a peptide defect within a triple helix.


Assuntos
Colágeno/química , Colágeno/isolamento & purificação , Peptídeos/química , Peptídeos/isolamento & purificação , Sequência de Aminoácidos , Varredura Diferencial de Calorimetria , Fracionamento Químico , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Dados de Sequência Molecular , Peso Molecular , Peptídeos/síntese química , Estabilidade Proteica , Estrutura Secundária de Proteína
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