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1.
J Biol Chem ; 300(5): 107293, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38636660

RESUMEN

Unsaturated fatty acid ketones with αß,γδ conjugation are susceptible to Michael addition of thiols, with unresolved issues on the site of adduction and precise structures of the conjugates. Herein we reacted 13-keto-octadecadienoic acid (13-oxo-ODE or 13-KODE) with glutathione (GSH), N-acetyl-cysteine, and ß-mercaptoethanol and identified the adducts. HPLC-UV analyses indicated none of the products exhibit a conjugated enone UV chromophore, a result that conflicts with the literature and is relevant to the mass spectral interpretation of 1,4 versus 1,6 thiol adduction. Aided by the development of an HPLC solvent system that separates the GSH diastereomers and thus avoids overlap of signals in proton NMR experiments, we established the two major conjugates are formed by 1,6 addition of GSH at the 9-carbon of 13-oxo-ODE with the remaining double bond α to the thiol in the 10,11 position. N-acetyl cysteine reacts similarly, while ß-mercaptoethanol gives equal amounts of 1,4 and 1,6 addition products. Equine glutathione transferase catalyzed 1,6 addition of GSH to the two major diastereomers in 44:56 proportions. LC-MS in positive ion mode gives a product ion interpreted before as evidence of 1,4-thiol adduction, whereas here we find this ion using the authentic 1,6 adduct. LC-MS with negative ion APCI gave a fragment selective for 1,4 adduction. These results clarify the structures of thiol conjugates of a prototypical unsaturated keto-fatty acid and have relevance to the application of LC-MS for the structural analysis of keto-fatty acid glutathione conjugation.


Asunto(s)
Glutatión , Compuestos de Sulfhidrilo , Glutatión/química , Glutatión/metabolismo , Compuestos de Sulfhidrilo/química , Espectrometría de Masas/métodos , Cromatografía Líquida de Alta Presión/métodos , Cromatografía Liquida/métodos , Mercaptoetanol/química , Cromatografía Líquida con Espectrometría de Masas
2.
J Biol Chem ; 300(4): 107173, 2024 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-38499149

RESUMEN

Sunlight exposure results in an inflammatory reaction of the skin commonly known as sunburn, which increases skin cancer risk. In particular, the ultraviolet B (UVB) component of sunlight induces inflammasome activation in keratinocytes to instigate the cutaneous inflammatory responses. Here, we explore the intracellular machinery that maintains skin homeostasis by suppressing UVB-induced inflammasome activation in human keratinocytes. We found that pharmacological inhibition of autophagy promoted UVB-induced NLRP3 inflammasome activation. Unexpectedly, however, gene silencing of Atg5 or Atg7, which are critical for conventional autophagy, had no effect, whereas gene silencing of Beclin1, which is essential not only for conventional autophagy but also for Atg5/Atg7-independent alternative autophagy, promoted UVB-induced inflammasome activation, indicating an involvement of alternative autophagy. We found that damaged mitochondria were highly accumulated in UVB-irradiated keratinocytes when alternative autophagy was inhibited, and they appear to be recognized by NLRP3. Overall, our findings indicate that alternative autophagy, rather than conventional autophagy, suppresses UVB-induced NLRP3 inflammasome activation through the clearance of damaged mitochondria in human keratinocytes and illustrate a previously unknown involvement of alternative autophagy in inflammation. Alternative autophagy may be a new therapeutic target for sunburn and associated cutaneous disorders.


Asunto(s)
Autofagia , Inflamasomas , Queratinocitos , Mitocondrias , Proteína con Dominio Pirina 3 de la Familia NLR , Rayos Ultravioleta , Humanos , Autofagia/efectos de la radiación , Proteína 5 Relacionada con la Autofagia/metabolismo , Proteína 5 Relacionada con la Autofagia/genética , Proteína 7 Relacionada con la Autofagia/genética , Proteína 7 Relacionada con la Autofagia/metabolismo , Beclina-1/metabolismo , Beclina-1/genética , Inflamasomas/metabolismo , Queratinocitos/metabolismo , Queratinocitos/patología , Queratinocitos/efectos de la radiación , Mitocondrias/metabolismo , Mitocondrias/efectos de la radiación , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Rayos Ultravioleta/efectos adversos , Células Cultivadas
3.
J Biol Chem ; 299(6): 104739, 2023 06.
Artículo en Inglés | MEDLINE | ID: mdl-37086788

RESUMEN

A key requirement in forming the water permeability barrier in the mammalian epidermis is the oxidation of linoleate esterified in a skin-specific acylceramide by the sequential actions of 12R-lipoxygenase, epidermal lipoxygenase-3, and the epoxyalcohol dehydrogenase SDR9C7 (short-chain dehydrogenase-reductase family 7 member 9). By mechanisms that remain unclear, this oxidation pathway promotes the covalent binding of ceramides to protein, forming a critical structure of the epidermal barrier, the corneocyte lipid envelope. Here, we detected, in porcine, mouse, and human epidermis, two novel fatty acid derivatives formed by KOH treatment from precursors covalently bound to protein: a "polar" lipid chromatographing on normal-phase HPLC just before omega-hydroxy ceramide and a "less polar" lipid nearer the solvent front. Approximately 100 µg of the novel lipids were isolated from porcine epidermis, and the structures were established by UV-spectroscopy, LC-MS, GC-MS, and NMR. Each is a C18 fatty acid and hydroxy-cyclohexenone with the ring on carbons C9-C14 in the polar lipid and C8-C13 in the less polar lipid. Overnight culture of [14C]linoleic acid with whole mouse skin ex vivo led to recovery of the 14C-labeled hydroxy-cyclohexenones. We deduce they are formed from covalently bound precursors during the KOH treatment used to release esterified lipids. KOH-induced intramolecular aldol reactions from a common precursor can account for their formation. Discovery of these hydroxy-cyclohexenones presents an opportunity for a reverse pathway analysis, namely to work back from these structures to identify their covalently bound precursors and relationship to the linoleate oxidation pathway.


Asunto(s)
Ceramidas , Epidermis , Ácido Linoleico , Lipooxigenasa , Animales , Humanos , Ratones , Ceramidas/metabolismo , Epidermis/metabolismo , Ácidos Grasos/metabolismo , Ácido Linoleico/metabolismo , Ácidos Linoleicos , Porcinos
5.
Free Radic Res ; 58(6-7): 430-438, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-39099129

RESUMEN

Heme-initiated decomposition of unsaturated fatty acid hydroperoxides creates alkoxyl radicals that propagate a complex series of reactions to hydroxy, keto, epoxy and aldehydic products. Herein, among the products from the hematin-catalyzed degradation of 9-hydroperoxy-linoleic acid (9-HPODE), we observed a double peak on normal-phase HPLC that resolved on RP-HPLC into equal proportions of two epoxy-allylic ketones with identical UV spectra. Their proton NMR spectra were also indistinguishable and consistent with 9,10-trans-epoxy-11E-13-keto- and 9-keto-10E-12,13-trans-epoxy-octadecenoic acids. Acid hydrolysis to the corresponding dihydroxy-ketones and GC-MS analysis identified the earlier eluting product on RP-HPLC as the 9,10-epoxy regio-isomer. Starting from the C9-hydroperoxide, recovery of the two epoxy-ketones in equal proportions suggests their formation from a common intermediate. Earlier work has proposed formation of a pseudo-symmetrical diepoxy radical (9,10-epoxy-11(•)-12,13-epoxy, derived from an epoxy allylic hydroperoxide precursor) in the carbon chain fragmentation leading to aldehydic products. This intermediate in pathways of alkoxyl radical reactions forms equal pairs of aldehydes, and now also a pair of epoxy-ketones, and based on mechanism the same products arise from either 9-HPODE or 13-HPODE. Our results point to the intermediacy of this diepoxy-carbinyl radical in the origin of at least two classes of linoleate peroxidation products, and it should be considered as a viable intermediate for homo-conjugated diene peroxidation in general. The reactions could contribute to the aldehydes and epoxy-ketones in tissues undergoing oxidative transformations of polyunsaturated fatty acids.


Asunto(s)
Compuestos Epoxi , Hemina , Cetonas , Ácido Linoleico , Peróxidos , Hemina/química , Peróxidos/química , Catálisis , Ácido Linoleico/química , Compuestos Epoxi/química , Cetonas/química , Radicales Libres/química , Estructura Molecular , Cromatografía Líquida de Alta Presión
6.
Artículo en Inglés | MEDLINE | ID: mdl-37336389

RESUMEN

ω-Alkynyl-fatty acids can be used as probes for covalent binding to intracellular macromolecules. To inform future in vivo studies, we determined the rates of reaction of ω-alkynyl-labeled linoleate with recombinant enzymes of the skin 12R-lipoxygenase (12R-LOX) pathway involved in epidermal barrier formation (12R-LOX, epidermal lipoxygenase-3 (eLOX3), and SDR9C7). We also examined the reactivity of ω-alkynyl-arachidonic acid with representative lipoxygenase enzymes employing either "carboxyl end-first" substrate binding (5S-LOX) or "tail-first" (platelet-type 12S-LOX). ω-Alkynyl-linoleic acid was oxygenated by 12R-LOX at 62 ± 9 % of the rate compared to linoleic acid, the alkynyl-9R-HPODE product was isomerized by eLOX3 at only 43 ± 1 % of the natural substrate, whereas its epoxy alcohol product was converted to epoxy ketone linoleic by an NADH-dependent dehydrogenase (SDR9C7) with 91 ± 1 % efficiency. The results suggest the optimal approach will be application of the 12R-LOX/eLOX3-derived epoxyalcohol, which should be most efficiently incorporated into the pathway and allow subsequent analysis of covalent binding to epidermal proteins. Regarding the orientation of substrate binding in LOX catalysis, our results and previous reports suggest the ω-alkynyl group has a stronger inhibitory effect on tail-first binding, as might be expected. Beyond slowing the reaction, however, we found that the tail-first binding and transformation of ω-alkynyl-arachidonic acid by platelet-type 12S-LOX results in almost complete enzyme inactivation, possibly due to reactive intermediates blocking the enzyme active site. Overall, the results reinforce the conclusion that ω-alkynyl-fatty acids are suitable for selected applications after appropriate reactivity is established.


Asunto(s)
Ácidos Araquidónicos , Piel , Piel/metabolismo , Lipooxigenasa/metabolismo , Ácido Linoleico/química , Ácidos Linoleicos/metabolismo , Ácidos Grasos , Ácido Araquidónico
7.
FEBS J ; 289(22): 7213-7220, 2022 11.
Artículo en Inglés | MEDLINE | ID: mdl-34787961

RESUMEN

We previously discovered an autophagy-like proteolysis mechanism that uses the Golgi membrane, namely, Golgi membrane-associated degradation (GOMED). Morphologically, GOMED resembles canonical autophagy, but the two mechanisms have different cellular functions, as they degrade different substrates and use different membrane sources. Furthermore, although the molecules involved partially overlap, the core molecules are completely different. GOMED preferentially degrades Golgi-trafficking proteins, including insulin granules in pancreatic ß-cells and ceruloplasmin in neurons, and is involved in a wide variety of physiological events.


Asunto(s)
Aparato de Golgi , Células Secretoras de Insulina , Aparato de Golgi/metabolismo , Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismo , Autofagia/fisiología , Proteolisis , Células Secretoras de Insulina/metabolismo
8.
Sci Rep ; 12(1): 22452, 2022 12 27.
Artículo en Inglés | MEDLINE | ID: mdl-36575188

RESUMEN

Autophagy results in the degradation of cytosolic components via two major membrane deformations. First, the isolation membrane sequesters components from the cytosol and forms autophagosomes, by which open structures become closed compartments. Second, the outer membrane of the autophagosomes fuses with lysosomes to degrade the inner membrane and its contents. The efficiency of the latter degradation process, namely autophagic flux, can be easily evaluated using lysosomal inhibitors, whereas the dynamics of the former process is difficult to analyze because of the challenges in identifying closed compartments of autophagy (autophagosomes and autolysosomes). To resolve this problem, we here developed a method to detect closed autophagic compartments by applying the FLIP technique, and named it FLIP-based Autophagy Detection (FLAD). This technique visualizes closed autophagic compartments and enables differentiation of open autophagic structures and closed autophagic compartments in live cells. In addition, FLAD analysis detects not only starvation-induced canonical autophagy but also genotoxic stress-induced alternative autophagy. By the combinational use of FLAD and LC3, we were able to distinguish the structures of canonical autophagy from those of alternative autophagy in a single cell.


Asunto(s)
Autofagosomas , Autofagia , Autofagosomas/metabolismo , Lisosomas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo
9.
Commun Biol ; 2: 37, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30701202

RESUMEN

Beclin 1 is a key regulator of autophagy and endocytosis. However, its autophagy-independent functions remain poorly understood. Here, we report that Beclin 1 regulates recycling endosome and is required for skin development in vivo. We first established keratinocyte-specific Beclin 1-knockout mice and found that these mutant mice died owing to severe impairment of epidermal barrier. Beclin 1 plays a role in autophagy and the endocytic pathway in cooperation with Atg14 and UVRAG, respectively, and keratinocyte-specific Atg14-knockout mice do not show any abnormal phenotypes, suggesting that Beclin 1 has a role in skin development via the endocytic pathway. Furthermore, we found that Beclin 1 deficiency causes mislocalization of integrins via a defect of recycling endosome, abnormal cell detachment of basal cells and their immature differentiation, and abnormal skin development. These results provide the first genetic evidence showing the roles of Beclin 1 in recycling endosome and skin development.


Asunto(s)
Beclina-1/genética , Endosomas/metabolismo , Organogénesis/genética , Piel/embriología , Piel/metabolismo , Animales , Autofagia/genética , Beclina-1/metabolismo , Diferenciación Celular , Células Cultivadas , Endocitosis/genética , Epidermis/embriología , Epidermis/metabolismo , Regulación del Desarrollo de la Expresión Génica , Humanos , Queratinocitos/citología , Queratinocitos/metabolismo , Ratones , Ratones Noqueados , Fenotipo
10.
J Nat Med ; 62(4): 479-80, 2008 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-18553179

RESUMEN

From dried whole plants of Glechoma hederacea L. (Labiatae), seven known glycosides were isolated and identified: (6R,7E,9R)-megastigma-4,7-dien-3-one 9-O-beta-D-glucopyranoside (1), apigenin 7-O-neohesperidoside (2), chrysoeriol 7-O-neohesperidoside (3), (+)-pinoresinol 4,4'-bis-O-beta-D-glucopyranoside (4), (+)-syringaresinol 4,4'-bis-O-beta-D-glucopyranoside (5), (+)-lariciresinol 4,4'-bis-O-beta-D-glucopyranoside (6), and (7R,8R)-threo-7,9,9'-trihydroxy-3,3'-dimethoxy-8-O-4'-neolignan 4-O-beta-D-glucopyranoside (7).


Asunto(s)
Glicósidos/aislamiento & purificación , Lamiaceae/química , Extractos Vegetales/química , Glicósidos/química , Plantas Medicinales/química
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