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Nearly one-third of nascent proteins are initially targeted to the endoplasmic reticulum (ER), where they are correctly folded and assembled before being delivered to their final cellular destinations. To prevent the accumulation of misfolded membrane proteins, ER-associated degradation (ERAD) removes these client proteins from the ER membrane to the cytosol in a process known as retrotranslocation. Our previous work demonstrated that rhomboid pseudoprotease Dfm1 is involved in the retrotranslocation of ubiquitinated membrane integral ERAD substrates. Herein, we found that Dfm1 associates with the SPOTS complex, which is composed of serine palmitoyltransferase (SPT) enzymes and accessory components that are critical for catalyzing the first rate-limiting step of the sphingolipid biosynthesis pathway. Furthermore, Dfm1 employs an ERAD-independent role for facilitating the ER export and endosome- and Golgi-associated degradation (EGAD) of Orm2, which is a major antagonist of SPT activity. Given that the accumulation of human Orm2 homologs, ORMDLs, is associated with various pathologies, our study serves as a molecular foothold for understanding how dysregulation of sphingolipid metabolism leads to various diseases.
Assuntos
Degradação Associada com o Retículo Endoplasmático , Esfingolipídeos , Humanos , Esfingolipídeos/metabolismo , Ubiquitina/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , HomeostaseRESUMO
The fatty acid (FA) elongation cycle produces very-long-chain FAs with ≥C21, which have unique physiological functions. Trans-2-enoyl-CoA reductases (yeast, Tsc13; mammals, TECR) catalyze the reduction reactions in the fourth step of the FA elongation cycle and in the sphingosine degradation pathway. However, their catalytic residues and coordinated action in the FA elongation cycle complex are unknown. To reveal these, we generated and analyzed Ala-substituted mutants of 15 residues of Tsc13. An in vitro FA elongation assay showed that nine of these mutants were less active than WT protein, with E91A and Y256A being the least active. Growth complementation analysis, measurement of ceramide levels, and deuterium-sphingosine labeling revealed that the function of the E91A mutant was substantially impaired in vivo. In addition, we found that the activity of FA elongases, which catalyze the first step of the FA elongation cycle, were reduced in the absence of Tsc13. Similar results were observed in Tsc13 E91A-expressing cells, which is attributable to reduced interaction between the Tsc13 E91A mutant and the FA elongases Elo2/Elo3. Finally, we found that E94A and Y248A mutants of human TECR, which correspond to E91A and Y256A mutants of Tsc13, showed reduced and almost no activity, respectively. Based on these results and the predicted three-dimensional structure of Tsc13, we speculate that Tyr256/Tyr248 of Tsc13/TECR is the catalytic residue that supplies a proton to trans-2-enoyl-CoAs. Our findings provide a clue concerning the catalytic mechanism of Tsc13/TECR and the coordinated action in the FA elongation cycle complex.
Assuntos
Ácidos Graxos Dessaturases , Esfingosina , Humanos , Ácidos Graxos Dessaturases/metabolismo , Elongases de Ácidos Graxos/metabolismo , Ácidos Graxos/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Esfingosina/metabolismoRESUMO
Maintaining the integrity of the nuclear envelope (NE) is essential for preventing genomic DNA damage. Recent studies have shown that enzymes that catalyze lipid synthesis are involved in NE maintenance, but the underlying mechanism remains unclear. Here, we found that the ceramide synthase (CerS) homolog in the fission yeast Schizosaccharomyces pombe Tlc4 (SPAC17A2.02c) suppressed NE defects in cells lacking the NE proteins Lem2 and Bqt4. Tlc4 possesses a TRAM/LAG1/CLN8 domain that is conserved in CerS proteins and functions through its non-catalytic activity. Tlc4 was localized at the NE and endoplasmic reticulum, similar to CerS proteins, and also showed unique additional localization at the cis- and medial-Golgi cisternae. Growth and mutation analyses revealed that Golgi localization of Tlc4 was tightly linked to its activity of suppressing the defects in the double-deletion mutant of Lem2 and Bqt4. Our results suggest that Lem2 and Bqt4 control the translocation of Tlc4 from the NE to the Golgi, which is necessary for maintaining NE integrity.
Assuntos
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Membrana Nuclear/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas Nucleares/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismoRESUMO
Lipids in the stratum corneum play an important role in the formation of the skin permeability barrier. The causative gene for congenital ichthyosis, NIPAL4, encodes a Mg2+ transporter and is involved in increases in intracellular Mg2+ concentrations that depend on keratinocyte differentiation. However, the role of this increased Mg2+ concentration in skin barrier formation and its effect on the lipid composition of the stratum corneum has remained largely unknown. Therefore, in the present study, we performed a detailed analysis of epidermal lipids in Nipal4 KO mice via TLC and MS. Compared with WT mice, the Nipal4 KO mice showed compositional changes in many ceramide classes (including decreases in ω-O-acylceramides and increases in ω-hydroxy ceramides), together with increases in ω-hydroxy glucosylceramides, triglycerides, and free fatty acids and decreases in ω-O-acyl hydroxy fatty acids containing a linoleic acid. We also found increases in unusual ω-O-acylceramides containing oleic acid or palmitic acid in the KO mice. However, there was little change in levels of cholesterol or protein-bound ceramides. The TLC analysis showed that some unidentified lipids were increased, and the MS analysis showed that these were special ceramides called 1-O-acylceramides. These results suggest that elevated Mg2+ concentrations in differentiated keratinocytes affect the production of various lipids, resulting in the lipid composition necessary for skin barrier formation.
Assuntos
Epiderme , Magnésio , Camundongos Knockout , Animais , Camundongos , Proteínas de Transporte de Cátions/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/deficiência , Ceramidas/metabolismo , Epiderme/metabolismo , Proteínas de Transporte de Ácido Graxo , Queratinócitos/metabolismo , Metabolismo dos Lipídeos , Lipídeos/análise , Magnésio/metabolismo , Receptores de Superfície Celular/metabolismoRESUMO
Ceramides are essential lipids for skin barrier function, and various classes and species exist in the human stratum corneum (SC). To date, the relationship between skin conditions and ceramide composition in healthy individuals has remained largely unclear. In the present study, we measured six skin condition parameters (capacitance, transepidermal water loss, scaliness, roughness, multilayer exfoliation, and corneocyte cell size) for the SC of the cheeks and upper arms of 26 healthy individuals and performed correlation analyses with their SC ceramide profiles, which we measured via liquid chromatography-tandem mass spectrometry. In the cheeks, high levels and/or ratios of two free ceramide classes containing an extra hydroxyl group in the long-chain moiety and a protein-bound ceramide class containing 6-hydroxysphingosine correlated with healthy skin conditions. In contrast, the ratios of two other free ceramide classes, both containing sphingosine, and a protein-bound ceramide class containing 4,14-sphingadiene correlated with unhealthy skin conditions, as did shortening of the carbon chain of the fatty acid portion of two ceramide classes containing non-hydroxy fatty acids. Thus, our findings help to elucidate the relationship between skin conditions and ceramide composition.
Assuntos
Ceramidas , Epiderme , Humanos , Ceramidas/metabolismo , Ceramidas/análise , Feminino , Masculino , Epiderme/metabolismo , Adulto , Pessoa de Meia-Idade , Pele/metabolismo , Voluntários Saudáveis , Espectrometria de Massas em Tandem , Adulto JovemRESUMO
BACKGROUND: This study aimed to investigate the prevalence of social frailty and associated factors. METHODS: A total of 655 consecutive patients who were able to complete the Kihon Checklist (KCL) and the Questionnaire on Social Frailty between June and August 2022 were enrolled. Social frailty was assessed using the Makizako Social Frailty Index. Patient characteristics were analyzed by analysis of variance. Factors associated with social frailty were analyzed using multivariate logistic analysis. Spearman's rank correlation coefficients were used to examine correlations between each KCL domain and social frailty. RESULTS: Mean age was 68 years, and disease duration was 12 years; 73% of patients were female. Social frailty was present in 30.8% of patients, with 36.5% classified as social pre-frailty. Multivariate analysis revealed age and HAQ-DI to be independent factors associated with social frailty. The proportion of social frailty increased with increasing age and worsening HAQ-DI scores. The KCL domain "Isolation" was the most strongly associated with social frailty (r = 0.601, P < 0.001), with higher scores associated with a higher proportion of social frailty. CONCLUSIONS: Social frailty in RA patients is associated with age and physical impairment (HAQ-DI). Moreover, the KCL domain "Isolation" was strongly associated with social frailty.
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OBJECTIVES: The present study aimed to examine discrepancies between assessments based on Routine Assessment of Patient Index Data 3 (RAPID3) and Simple Disease Activity Index (SDAI) in RA patients with controlled disease activity. METHODS: Data from 464 RA patients in SDAI remission or low disease activity (REM/LDA) were analyzed. Patient-reported outcome (PRO) measures, including Health Assessment Questionnaire Disability Index (HAQ-DI), 25-question Geriatric Locomotive Function Scale (GLFS-25), and Kihon checklist (KCL), were assessed. Logistic regression models were used to identify factors associated with RAPID3 moderate or high disease activity (MDA/HDA). Cutoff values of RAPID3 MDA/HDA for each PRO evaluation item were determined using receiver operating characteristic curve analysis. RESULTS: Among RA patients in SDAI REM/LDA, 84.9% were in RAPID3 REM/LDA. Multivariable analysis revealed that HAQ-DI, GLFS-25, and KCL were independently associated with RAPID3 MDA/HDA. Subdomain analysis of KCL revealed that activities of daily living, physical function, cognitive function, and depressive mood were significantly associated with RAPID3 MDA/HDA. Cutoff values for HAQ-DI and KCL were 0.38 and 8, respectively. CONCLUSIONS: In RA patients with controlled disease activity, discrepancies between RAPID3 and SDAI assessments were observed, with factors such as HAQ-DI, GLFS-25, and KCL being independently associated with RAPID3 MDA/HDA.
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The epidermis-specific lipid acylceramide plays a pivotal role in the formation of the permeability barrier in the skin; abrogation of its synthesis causes the skin disorder ichthyosis. However, the acylceramide synthetic pathway has not yet been fully elucidated: Namely, the acyl-CoA synthetase (ACS) involved in this pathway remains to be identified. Here, we hypothesized it to be encoded by FATP4/ACSVL4, the causative gene of ichthyosis prematurity syndrome (IPS). In vitro experiments revealed that FATP4 exhibits ACS activity toward an ω-hydroxy fatty acid (FA), an intermediate of the acylceramide synthetic pathway. Fatp4 knockout (KO) mice exhibited severe skin barrier dysfunction and morphological abnormalities in the epidermis. The total amount of acylceramide in Fatp4 KO mice was reduced to â¼10% of wild-type mice. Decreased levels and shortening of chain lengths were observed in the saturated, nonacylated ceramides. FA levels were not decreased in the epidermis of Fatp4 KO mice. The expression levels of the FA elongase Elovl1 were reduced in Fatp4 KO epidermis, partly accounting for the reduction and shortening of saturated, nonacylated ceramides. A decrease in acylceramide levels was also observed in human keratinocytes with FATP4 knockdown. From these results, we conclude that skin barrier dysfunction observed in IPS patients and Fatp4 KO mice is caused mainly by reduced acylceramide production. Our findings further elucidate the molecular mechanism governing acylceramide synthesis and IPS pathology.
Assuntos
Ceramidas/metabolismo , Epiderme/metabolismo , Proteínas de Transporte de Ácido Graxo/metabolismo , Ictiose/metabolismo , Doenças do Prematuro/metabolismo , Animais , Ceramidas/química , Proteínas de Transporte de Ácido Graxo/genética , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Feminino , Humanos , Ictiose/genética , Doenças do Prematuro/genética , Masculino , Camundongos Knockout , Permeabilidade , Pele/metabolismoRESUMO
Ceramides are essential lipids for skin permeability barrier function, and a wide variety of ceramide species exist in the stratum corneum (SC). Although ceramides with long-chain bases (LCBs) of various lengths have been identified in the human SC, a quantitative analysis that distinguishes ceramide species with different LCB chain lengths has not been yet published. Therefore, the whole picture of human SC ceramides remains unclear. Here, we conducted LC/MS/MS analyses to detect individual ceramide species differing in both the LCB and FA chain lengths and quantified 1,327 unbound ceramides and 254 protein-bound ceramides: the largest number of ceramide species reported to date. Ceramides containing an LCB whose chain length was C16-26 were present in the human SC. Of these, C18 (28.6%) was the most abundant, followed by C20 (24.8%) and C22 (12.8%). Each ceramide class had a characteristic distribution of LCB chain lengths and was divided into five groups according to this distribution. There was almost no difference in FA composition between the ceramide species containing LCBs of different chain lengths. Furthermore, we demonstrated that one of the serine palmitoyltransferase (SPT) complexes, SPTLC1/SPTLC3/SPTSSB, was able to produce C16-24 LCBs. The expression levels of all subunits constituting the SPT complexes increased during keratinocyte differentiation, resulting in the observed chain-length diversity of LCBs in the human SC. This study provides a molecular basis for elucidating human SC ceramide diversity and the pathogenesis of skin disorders.
Assuntos
Ceramidas , Espectrometria de Massas em Tandem , Ceramidas/metabolismo , Epiderme/metabolismo , Humanos , Permeabilidade , Serina C-Palmitoiltransferase/genética , Serina C-Palmitoiltransferase/metabolismoRESUMO
Self-healing collodion baby (SHCB), also called "self-improving collodion baby", is a rare mild variant of autosomal recessive congenital ichthyosis and is defined as a collodion baby who shows the nearly complete resolution of scaling within the first 3 months to 1 year of life. However, during the neonatal period, it is not easy to distinguish SHCB from other inflammatory forms of autosomal recessive congenital ichthyosis, such as congenital ichthyosiform erythroderma. Here, we report a case study of two Japanese SHCB patients with compound heterozygous mutations, c.235G>T (p.(Glu79∗))/ c.1189C>T (p.(Arg397Cys)) and c.1295A>G (p.(Tyr432Cys))/ c.1138delG (p.(Asp380Thrfs∗3)), in CYP4F22, which encodes cytochrome P450, family 4, subfamily F, polypeptide 22 (CYP4F22). Immunohistochemically, inflammation with the strong expression of IL-17C, IL-36γ, and TNF-α was seen in the skin at birth. CYP4F22 is an ultra-long-chain FA ω-hydroxylase responsible for ω-O-acylceramide (acylceramide) production. Among the epidermal ceramides, acylceramide is a key lipid in maintaining the epidermal permeability barrier function. We found that the levels of ceramides with ω-hydroxy FAs including acylceramides and the levels of protein-bound ceramides were much lower in stratum corneum samples obtained by tape stripping from SHCB patients than in those from their unaffected parents and individuals without SHCB. Additionally, our cell-based enzyme assay revealed that two mutants, p.(Glu79∗) and p.(Arg397Cys), had no enzyme activity. Our findings suggest that genetic testing coupled with noninvasive ceramide analyses using tape-stripped stratum corneum samples might be useful for the early and precise diagnosis of congenital ichthyoses, including SHCB.
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Ceramidas , Ictiose Lamelar , Lactente , Recém-Nascido , Humanos , Colódio , Ceramidas/metabolismo , Ictiose Lamelar/diagnóstico , Ictiose Lamelar/genética , Testes GenéticosRESUMO
T cell stimulation by bacterial superantigens induces a cytokine storm. After T cell activation and inflammatory cytokine secretion, regulatory T cells (Treg) are produced to suppress the immune response. Coccomyxa sp.KJ (IPOD FERM BP-22254), a green alga, is reported to regulate immune reactions. Therefore, we examined the effects of Coccomyxa sp.KJ extract (CE) on the superantigen-induced immune response. When human peripheral blood mononuclear cells (PBMCs) were stimulated with toxic shock syndrome-1 (TSST-1) in the presence of CE, the number of activated T cells decreased moderately. Purified T cells stimulated in the presence of CE comprised more non-proliferating cells than those stimulated in the absence of CE, whereas some T cells proliferated more quickly. The levels of activation markers on the stimulated T cells increased in the presence of CE. Most of the inflammatory cytokines did not change but IL-1ß, IL-17, IL-4, and IL-13 secretion increased, whereas that of IL-2, TNF-α, and IL-18 decreased. IL-10 secretion was also decreased by CE treatment, suggesting that the immune response was not suppressed by Treg cells. CE enhanced the expression of stem cell-like memory cell markers in T cells. These results suggest that CE can regulate the fate of T cells and can help to ameliorate superantigen-induced T cell hyperactivation and immune suppression.
Assuntos
Clorófitas , Infecções Estafilocócicas , Toxinas Bacterianas , Enterotoxinas , Humanos , Leucócitos Mononucleares , Ativação Linfocitária , Staphylococcus aureus , Superantígenos/metabolismoRESUMO
In eukaryotic cells, chromosomes are confined to the nucleus, which is compartmentalized by the nuclear membranes; these are continuous with the endoplasmic reticulum membranes. Maintaining the homeostasis of these membranes is an important cellular activity performed by lipid metabolic enzymes. However, how lipid metabolic enzymes affect nuclear membrane functions remains to be elucidated. We found that the very-long-chain fatty acid elongase Elo2 is located in the nuclear membrane and prevents lethal defects associated with nuclear membrane ruptures in mutants of the nuclear membrane proteins Lem2 and Bqt4 in the fission yeast Schizosaccharomyces pombe. Lipid composition analysis shows that t20:0/24:0 phytoceramide (a conjugate of C20:0 phytosphingosine and C24:0 fatty acid) is a major ceramide species in S. pombe The quantity of this ceramide is reduced in the absence of Lem2, and restored by increased expression of Elo2. Furthermore, loss of S. pombe Elo2 can be rescued by its human orthologs. These results suggest that the conserved very-long-chain fatty acid elongase producing the ceramide component is essential for nuclear membrane integrity and cell viability in eukaryotes.This article has an associated First Person interview with the first author of the paper.
Assuntos
Acetiltransferases/metabolismo , Elongases de Ácidos Graxos/metabolismo , Proteínas de Membrana/metabolismo , Membrana Nuclear/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Humanos , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Over the last two decades, extensive studies have been performed at the molecular level to understand the evolution of carnivorous plants. As fruits, the repertoire of protein components in the digestive fluids of several carnivorous plants have gradually become clear. However, the quantitative aspects of these proteins and the expression mechanisms of the genes that encode them are still poorly understood. In this study, using the Australian sundew Drosera adelae, we identified and quantified the digestive fluid proteins. We examined the expression and methylation status of the genes corresponding to major hydrolytic enzymes in various organs; these included thaumatin-like protein, S-like RNase, cysteine protease, class I chitinase, ß-1, 3-glucanase, and hevein-like protein. The genes encoding these proteins were exclusively expressed in the glandular tentacles. Furthermore, the promoters of the ß-1, 3-glucanase and cysteine protease genes were demethylated only in the glandular tentacles, similar to the previously reported case of the S-like RNase gene da-I. This phenomenon correlated with high expression of the DNA demethylase DEMETER in the glandular tentacles, strongly suggesting that it performs glandular tentacle-specific demethylation of the genes. The current study strengthens and generalizes the relevance of epigenetics to trap organ-specific gene expression in D. adelae. We also suggest similarities between the trap organs of carnivorous plants and the roots of non-carnivorous plants.
Assuntos
Drosera , Epigênese Genética , Austrália , Drosera/enzimologia , Drosera/genética , Folhas de Planta , Proteínas de Plantas/genética , Ribonucleases/genéticaRESUMO
Sphingolipids are multifunctional lipids. Among the sphingolipid-component sphingoid bases, 4,14-sphingadiene (SPD) is unique such that it has a cis double bond with a bent structure. Although SPD was discovered half a century ago, its tissue distribution, biosynthesis, and degradation remain poorly understood. Here, we established a specific and quantitative method for SPD measurement and found that SPD exists in a wide range of mammalian tissues. SPD was especially abundant in kidney, where the amount of SPD was ~2/3 of sphingosine, the most abundant sphingoid base in mammals. Although SPD is metabolized to ceramides and SPD 1-phosphate with almost the same efficiency as sphingosine, it is less susceptible to degradation by a cleavage reaction, at least in vitro. We identified the fatty acid desaturase family protein FADS3 as a ceramide desaturase that produces SPD ceramides by desaturating ceramides containing sphingosine. SPD sphingolipids were preferentially localized outside lipid microdomains, suggesting that SPD has different functions compared to other sphingoid bases in the formation of lipid microdomains. In summary, we revealed the biosynthesis and degradation pathways of SPD and its characteristic membrane localization. Our findings contribute to the elucidation of the molecular mechanism underlying the generation of sphingolipid diversity.
Assuntos
Ceramidas/metabolismo , Ácidos Graxos Dessaturases/metabolismo , Esfingosina/metabolismo , Animais , Ácidos Graxos Dessaturases/genética , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Microdomínios da Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Esfingosina/análogos & derivadosRESUMO
Ceramides are the predominant lipids in the stratum corneum (SC) and are crucial components for normal skin barrier function. Although the composition of various ceramide classes in the human SC has been reported, that in mice is still unknown, despite mice being widely used as animal models of skin barrier function. Here, we performed LC/MS/MS analyses using recently available ceramide class standards to measure 25 classes of free ceramides and 5 classes of protein-bound ceramides from human and mouse SC. Phytosphingosine- and 6-hydroxy sphingosine-type ceramides, which both contain an additional hydroxyl group, were abundant in the human SC (35% and 45% of total ceramides, respectively). In contrast, in mice, phytosph-ingosine- and 6-hydroxy sphingosine-type ceramides were present at â¼1% and undetectable levels, respectively, and sphingosine-type ceramides accounted for â¼90%. In humans, ceramides containing α-hydroxy FA were abundant, whereas ceramides containing ß-hydroxy or ω-hydroxy FA were abundant in mice. The hydroxylated ß-carbon in ß-hydroxy ceramides was in the (R) configuration. Genetic knockout of ß-hydroxy acyl-CoA dehydratases in HAP1 cells increased ß-hydroxy ceramide levels, suggesting that ß-hydroxy acyl-CoA, an FA-elongation cycle intermediate in the ER, is a substrate for ß-hydroxy ceramide synthesis. We anticipate that our methods and findings will help to elucidate the role of each ceramide class in skin barrier formation and in the pathogenesis of skin disorders.
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Ceramidas/metabolismo , Cromatografia Líquida , Epiderme/metabolismo , Lipidômica/métodos , Espectrometria de Massas em Tandem , Animais , Humanos , CamundongosRESUMO
Sphingosine 1-phosphate (S1P) functions not only as a bioactive lipid molecule, but also as an important intermediate of the sole sphingolipid-to-glycerolipid metabolic pathway. However, the precise reactions and the enzymes involved in this pathway remain unresolved. We report here that yeast HFD1 and the Sjögren-Larsson syndrome (SLS)-causative mammalian gene ALDH3A2 are responsible for conversion of the S1P degradation product hexadecenal to hexadecenoic acid. The absence of ALDH3A2 in CHO-K1 mutant cells caused abnormal metabolism of S1P/hexadecenal to ether-linked glycerolipids. Moreover, we demonstrate that yeast Faa1 and Faa4 and mammalian ACSL family members are acyl-CoA synthetases involved in the sphingolipid-to-glycerolipid metabolic pathway and that hexadecenoic acid accumulates in Δfaa1 Δfaa4 mutant cells. These results unveil the entire S1P metabolic pathway: S1P is metabolized to glycerolipids via hexadecenal, hexadecenoic acid, hexadecenoyl-CoA, and palmitoyl-CoA. From our results we propose a possibility that accumulation of the S1P metabolite hexadecenal contributes to the pathogenesis of SLS.
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Aldeído Oxirredutases/genética , Aldeído Oxirredutases/metabolismo , Lisofosfolipídeos/metabolismo , Síndrome de Sjogren-Larsson/genética , Síndrome de Sjogren-Larsson/metabolismo , Esfingosina/análogos & derivados , Acil Coenzima A/genética , Acil Coenzima A/metabolismo , Animais , Células CHO , Coenzima A Ligases/genética , Coenzima A Ligases/metabolismo , Cricetinae , Cricetulus , Genes Fúngicos , Humanos , Redes e Vias Metabólicas , Mutação , Ácidos Palmíticos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Síndrome de Sjogren-Larsson/etiologia , Esfingosina/metabolismoRESUMO
Peptide vaccination was developed for the prevention and therapy of acute and chronic infectious diseases and cancer. However, vaccine development is challenging, because the patient immune system requires the appropriate human leukocyte antigen (HLA) recognition with the peptide. Moreover, antigens sometimes induce a low response, even if the peptide is presented by antigen-presenting cells and T cells recognize it. This is because the patient immunity is dampened or restricted by environmental factors. Even if the immune system responds appropriately, newly-developed immune checkpoint inhibitors (ICIs), which are used to increase the immune response against cancer, make the immune environment more complex. The ICIs may activate T cells, although the ratio of responsive patients is not high. However, the vaccine may induce some immune adverse effects in the presence of ICIs. Therefore, a system is needed to predict such risks. Humanized mouse systems possessing human immune cells have been developed to examine human immunity in vivo. One of the systems which uses transplanted human peripheral blood mononuclear cells (PBMCs) may become a new diagnosis strategy. Various humanized mouse systems are being developed and will become good tools for the prediction of antibody response and immune adverse effects.
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Imunoterapia , Vacinas de Subunidades Antigênicas/imunologia , Animais , Formação de Anticorpos , Humanos , Sistema Imunitário/metabolismo , Terapia de Imunossupressão , Camundongos , Modelos AnimaisRESUMO
Differences among fatty acids (FAs) in chain length and number of double bonds create lipid diversity. FA elongation proceeds via a four-step reaction cycle, in which the 3-hydroxyacyl-CoA dehydratases (HACDs) HACD1-4 catalyze the third step. However, the contribution of each HACD to 3-hydroxyacyl-CoA dehydratase activity in certain tissues or in different FA elongation pathways remains unclear. HACD1 is specifically expressed in muscles and is a myopathy-causative gene. Here, we generated Hacd1 KO mice and observed that these mice had reduced body and skeletal muscle weights. In skeletal muscle, HACD1 mRNA expression was by far the highest among the HACDs However, we observed only an â¼40% reduction in HACD activity and no changes in membrane lipid composition in Hacd1-KO skeletal muscle, suggesting that some HACD activities are redundant. Moreover, when expressed in yeast, both HACD1 and HACD2 participated in saturated and monounsaturated FA elongation pathways. Disruption of HACD2 in the haploid human cell line HAP1 significantly reduced FA elongation activities toward both saturated and unsaturated FAs, and HACD1 HACD2 double disruption resulted in a further reduction. Overexpressed HACD3 exhibited weak activity in saturated and monounsaturated FA elongation pathways, and no activity was detected for HACD4. We therefore conclude that HACD1 and HACD2 exhibit redundant activities in a wide range of FA elongation pathways, including those for saturated to polyunsaturated FAs, with HACD2 being the major 3-hydroxyacyl-CoA dehydratase. Our findings are important for furthering the understanding of the molecular mechanisms in FA elongation and diversity.
Assuntos
Ácidos Graxos/metabolismo , Hidroliases/metabolismo , Proteínas de Membrana/metabolismo , Músculo Esquelético/enzimologia , Mioblastos Esqueléticos/enzimologia , Proteínas Tirosina Fosfatases/metabolismo , Animais , Sistemas CRISPR-Cas , Domínio Catalítico , Linhagem Celular Tumoral , Células Cultivadas , Ácidos Graxos/química , Regulação Enzimológica da Expressão Gênica , Humanos , Hidroliases/genética , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Proteínas de Membrana/genética , Camundongos Knockout , Estrutura Molecular , Peso Molecular , Músculo Esquelético/citologia , Músculo Esquelético/patologia , Doenças Musculares/enzimologia , Doenças Musculares/genética , Doenças Musculares/patologia , Mioblastos Esqueléticos/citologia , Mioblastos Esqueléticos/patologia , Proteínas Tirosina Fosfatases/genética , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por SubstratoAssuntos
Antiasmáticos , Asma , Humanos , Camundongos , Animais , Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Asma/tratamento farmacológico , Imunoglobulina E/uso terapêutico , Antiasmáticos/uso terapêutico , Eosinófilos , Progressão da DoençaRESUMO
A skin permeability barrier is essential for terrestrial animals, and its impairment causes several cutaneous disorders such as ichthyosis and atopic dermatitis. Although acylceramide is an important lipid for the skin permeability barrier, details of its production have yet to be determined, leaving the molecular mechanism of skin permeability barrier formation unclear. Here we identified the cytochrome P450 gene CYP4F22 (cytochrome P450, family 4, subfamily F, polypeptide 22) as the long-sought fatty acid ω-hydroxylase gene required for acylceramide production. CYP4F22 has been identified as one of the autosomal recessive congenital ichthyosis-causative genes. Ichthyosis-mutant proteins exhibited reduced enzyme activity, indicating correlation between activity and pathology. Furthermore, lipid analysis of a patient with ichthyosis showed a drastic decrease in acylceramide production. We determined that CYP4F22 was a type I membrane protein that locates in the endoplasmic reticulum (ER), suggesting that the ω-hydroxylation occurs on the cytoplasmic side of the ER. The preferred substrate of the CYP4F22 was fatty acids with a carbon chain length of 28 or more (≥C28). In conclusion, our findings demonstrate that CYP4F22 is an ultra-long-chain fatty acid ω-hydroxylase responsible for acylceramide production and provide important insights into the molecular mechanisms of skin permeability barrier formation. Furthermore, based on the results obtained here, we proposed a detailed reaction series for acylceramide production.