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1.
Sci Adv ; 10(11): eadj6406, 2024 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-38489355

RESUMO

There is a compelling need to find drugs active against Mycobacterium tuberculosis (Mtb). 4'-Phosphopantetheinyl transferase (PptT) is an essential enzyme in Mtb that has attracted interest as a potential drug target. We optimized a PptT assay, used it to screen 422,740 compounds, and identified raltitrexed, an antineoplastic antimetabolite, as the most potent PptT inhibitor yet reported. While trying unsuccessfully to improve raltitrexed's ability to kill Mtb and remove its ability to kill human cells, we learned three lessons that may help others developing antibiotics. First, binding of raltitrexed substantially changed the configuration of the PptT active site, complicating molecular modeling of analogs based on the unliganded crystal structure or the structure of cocrystals with inhibitors of another class. Second, minor changes in the raltitrexed molecule changed its target in Mtb from PptT to dihydrofolate reductase (DHFR). Third, the structure-activity relationship for over 800 raltitrexed analogs only became interpretable when we quantified and characterized the compounds' intrabacterial accumulation and transformation.


Assuntos
Mycobacterium tuberculosis , Neoplasias , Quinazolinas , Tiofenos , Transferases (Outros Grupos de Fosfato Substituídos) , Humanos , Mycobacterium tuberculosis/metabolismo , Timidilato Sintase/metabolismo , Proteínas de Bactérias/metabolismo
2.
Cancer Immunol Immunother ; 73(3): 47, 2024 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-38349411

RESUMO

The response rate of anti-PD1 therapy is limited, and the influence of anti-PD1 therapy on cancer patients is unclear. To address these challenges, we conducted a longitudinal analysis of plasma proteomic changes with anti-PD1 therapy in non-small cell lung cancer (NSCLC), alveolar soft part sarcoma (ASPS), and lymphoma patients. We included 339 plasma samples before and after anti-PD1 therapy from 193 patients with NSCLC, ASPS, or lymphoma. The plasma proteins were detected using data-independent acquisition-mass spectrometry and customable antibody microarrays. Differential proteomic characteristics in responders (R) and non-responders (NR) before and after anti-PD1 therapy were elucidated. A total of 1019 proteins were detected using our in-depth proteomics platform and distributed across 10-12 orders of abundance. By comparing the differential plasma proteome expression between R and NR groups, 50, 206, and 268 proteins were identified in NSCLC, ASPS, and lymphoma patients, respectively. Th17, IL-17, and JAK-STAT signal pathways were identified upregulated in NR group, while cellular senescence and transcriptional misregulation pathways were activated in R group. Longitudinal proteomics analysis revealed the IL-17 signaling pathway was downregulated after treatment. Consistently, many proteins were identified as potential combinatorial therapeutic targets (e.g., IL-17A and CD22). Five noninvasive biomarkers (FLT4, SFTPB, GNPTG, F5, and IL-17A) were further validated in an independent lymphoma cohort (n = 39), and another three noninvasive biomarkers (KIT, CCL3, and TNFSF1) were validated in NSCLC cohort (n = 76). Our results provide molecular insights into the anti-PD1 therapy in cancer patients and identify new therapeutic strategies for anti-PD1-resistant patients.


Assuntos
Anti-Infecciosos , Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Linfoma , Humanos , Interleucina-17 , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Proteômica , Neoplasias Pulmonares/tratamento farmacológico , Penicilinas , Biomarcadores , Transferases (Outros Grupos de Fosfato Substituídos)
3.
J Cancer Res Clin Oncol ; 150(2): 46, 2024 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-38285090

RESUMO

OBJECTIVES: Multi-drug resistance (MDR) to chemotherapy is the main obstacle influencing the anti-tumor effect in breast cancer, which might lead to the metastasis and recurrence of cancer. Until now, there are still no effective methods that can overcome MDR. In this study, we aimed to investigate the role of sphingomyelin synthase 2 (SMS2) in breast cancer resistance. METHODS: Quantitative RT-PCR analysis was performed to assess changes in mRNA expression. Western blot analysis was performed to detect protein expression. Inhibitory concentration value of adriamycin (ADR) was evaluated using CCK 8 assay. The stemness ability of breast cancer cells was assessed by spheroid-formation assay. Immunofluorescence staining was conducted to show the cellular distribution of proteins. Breast tumor masses were harvested from the xenograft tumor mouse model. RESULTS: SMS2 overexpression increased the IC50 values of breast cancer cells. SMS2 decreased the CD24 transcription level but increased the transcription levels of stemness-related genes including CD44, ALDH, OCT 4 and SOX2 in breast cancer cells. SMS2 overexpression promoted the nuclear translocation of phosphorylated NF-κB, while suppression of SMS2 could inhibit the NF-κB pathway. CONCLUSIONS: SMS2 increased the stemness of breast cancer cells via NF-κB signaling pathway, leading to resistance to the chemotherapeutic drug ADR. Thus, SMS2 might play a critical role in the development of breast cancer resistance, which is a previously unrecognized mechanism in breast cancer MDR development.


Assuntos
Neoplasias da Mama , NF-kappa B , Transferases (Outros Grupos de Fosfato Substituídos) , Animais , Feminino , Humanos , Camundongos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Modelos Animais de Doenças , Doxorrubicina , Transdução de Sinais , Células-Tronco Neoplásicas
4.
J Med Genet ; 61(2): 125-131, 2024 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-37399314

RESUMO

BACKGROUND: Mitral annular disjunction (MAD) is an under-recognised phenotype associated with severe ventricular arrhythmias. Limited knowledge has been gained on its molecular genesis. METHODS: A total of 150 unrelated deceased Chinese were collected for whole-exome sequencing, with analysis focusing on a panel of 118 genes associated with 'abnormal mitral valve morphology'. Cases were prespecified as 'longitudinally extensive MAD (LE-MAD)' or 'longitudinally less-extensive MAD (LLE-MAD)' according to the gross disjunctional length with a cut-off of 4.0 mm. The pedigree investigation was conducted on a case carrying an ultra-rare (minor allele frequency <0.1%) deleterious variant in DCHS1. RESULTS: Seventy-seven ultra-rare deleterious variants were finally identified. Exclusively, 12 ultra-rare deleterious variants distributed in nine genes occurred in LE-MAD, which were ANK1, COL3A1, DCHS1, FBN2, GNPTAB, LZTR1, PLD1, RYR1 and VPS13B. Ultra-rare deleterious variants in those nine genes were predominantly distributed in LE-MAD compared with LLE-MAD (28% vs 5%, OR 7.30, 95% CI 2.33 to 23.38; p<0.001), and the only gene related to LE-MAD with borderline significance was DCHS1. LE-MAD was consistently observed in a sizeable Chinese family, in which LE-MAD independently co-segregated with an ultra-rare deleterious variant in DCHS1, rs145429962. CONCLUSION: This study initially proposed that isolated LE-MAD might be a particular phenotype of MAD with a complex genetic predisposition. Deleterious variants in DCHS1 might be associated with the morphogenesis of LE-MAD.


Assuntos
Doenças das Valvas Cardíacas , Prolapso da Valva Mitral , Humanos , Prolapso da Valva Mitral/genética , Valva Mitral , Mutação/genética , Arritmias Cardíacas , Suscetibilidade a Doenças , Fatores de Transcrição/genética , Transferases (Outros Grupos de Fosfato Substituídos)/genética
5.
Technol Cancer Res Treat ; 22: 15330338231212071, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37926998

RESUMO

PURPOSE: The malignant transformation of cells can lead to aerobic glycolysis, an important form of metabolic reprogramming in colon cancer cells, which can cause the accumulation of lactate and accelerate the proliferation of tumor cells also enhance their chemotherapy drug resistance. The aim of this study was to investigate the possible molecular mechanisms responsible for the increased lactate expression in colon cancer. METHODS: Several bioinformatics methods, including differential analysis, gene ontology enrichment, univariate and multivariate Cox regression analysis were used to find the lactic acid-related gene carnitine palmitoyltransferase 2. We analyzed the relationship between carnitine palmitoyltransferase 2 and clinical features as well as immune microenvironment. To further explore the mechanism of carnitine palmitoyltransferase 2 in colon cancer, we performed methylation analysis and constructed a competitive endogenous RNA network, which was validated in cell lines and clinical specimens. RESULTS: We used bioinformatics to select the lactic acid-related gene carnitine palmitoyltransferase 2 and found low expression of carnitine palmitoyltransferase 2 was associated with poor prognosis in colon cancer. An inhibitory tumor microenvironment was created when carnitine palmitoyltransferase 2 expression was reduced, with decreased CD4 T cells, CD8 T cells, dendritic cells, and B cells but increased cancer-associated fibroblasts. Methylation analysis showed that the abnormal decrease in carnitine palmitoyltransferase 2 might be caused by hypermethylation. We constructed a network of SGMS1-AS1/microRNA-106a-5p/carnitine palmitoyltransferase 2 and verified their expression in cell lines and clinical specimens. CONCLUSION: Our work revealed the possible mechanism of lactate accumulation in colon cancer and explored a new potential treatment for colon cancer by cutting off aerobic glycolysis in tumor cells.


Assuntos
Carnitina O-Palmitoiltransferase , Neoplasias do Colo , MicroRNAs , Humanos , Carnitina O-Palmitoiltransferase/genética , Carnitina O-Palmitoiltransferase/metabolismo , Transformação Celular Neoplásica , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Ácido Láctico , Proteínas de Membrana , MicroRNAs/genética , MicroRNAs/metabolismo , Proteínas do Tecido Nervoso , Transferases (Outros Grupos de Fosfato Substituídos) , Microambiente Tumoral/genética
6.
Front Endocrinol (Lausanne) ; 14: 1224318, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37886644

RESUMO

Pathogenic heterozygous variants in SGMS2 cause a rare monogenic form of osteoporosis known as calvarial doughnut lesions with bone fragility (CDL). The clinical presentations of SGMS2-related bone pathology range from childhood-onset osteoporosis with low bone mineral density and sclerotic doughnut-shaped lesions in the skull to a severe spondylometaphyseal dysplasia with neonatal fractures, long-bone deformities, and short stature. In addition, neurological manifestations occur in some patients. SGMS2 encodes sphingomyelin synthase 2 (SMS2), an enzyme involved in the production of sphingomyelin (SM). This review describes the biochemical structure of SM, SM metabolism, and their molecular actions in skeletal and neural tissue. We postulate how disrupted SM gradient can influence bone formation and how animal models may facilitate a better understanding of SGMS2-related osteoporosis.


Assuntos
Nervo Facial , Osteoporose , Transferases (Outros Grupos de Fosfato Substituídos) , Animais , Criança , Humanos , Recém-Nascido , Nervo Facial/metabolismo , Nervo Facial/patologia , Osteoporose/complicações , Osteoporose/patologia , Paralisia , Crânio/metabolismo , Esfingomielinas/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo
7.
FEBS Lett ; 597(21): 2672-2686, 2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37715942

RESUMO

Sphingomyelin (SM) synthase 1 (SMS1), which is involved in lipodystrophy, deafness, and thrombasthenia, generates diacylglycerol (DG) and SM using phosphatidylcholine (PC) and ceramide as substrates. Here, we found that SMS1 possesses DG-generating activities via hydrolysis of PC and phosphatidylethanolamine (PE) in the absence of ceramide and ceramide phosphoethanolamine synthase (CPES) activity. In the presence of the same concentration (4.7 mol%) of PC and ceramide, the amounts of DG produced by SMS and PC-phospholipase C (PLC) activities of SMS1 were approximately 65% and 35% of total DG production, respectively. PC-PLC activity showed substrate selectivity for saturated and/or monounsaturated fatty acid-containing PC species. A PC-PLC/SMS inhibitor, D609, inhibited only SMS activity. Mn2+ inhibited only PC-PLC activity. Intriguingly, DG attenuated SMS/CPES activities. Our study indicates that SMS1 is a unique enzyme with PC-PLC/PE-PLC/SMS/CPES activities.


Assuntos
Ceramidas , Esfingomielinas , Humanos , Diglicerídeos , Fosfatidilcolinas , Transferases (Outros Grupos de Fosfato Substituídos)/genética
8.
BMC Ophthalmol ; 23(1): 394, 2023 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-37752499

RESUMO

BACKGROUND: We describe the case of a 47-year-old man referred to a retinal clinic and diagnosed with late-onset retinitis pigmentosa. Surprisingly, genetic testing revealed compound heterozygous pathogenic variants in GNPTG, leading to the diagnosis of the autosomal recessive lysosomal storage disorder mucolipidosis type III gamma. Mucolipidosis type III gamma is typically diagnosed during childhood due to symptoms relating to skeletal dysplasia. Retinal dystrophy is not a common phenotypic feature. CASE PRESENTATION: Ophthalmologic examination was consistent with a mild form of retinitis pigmentosa and included fundus photography, measurement of best-corrected visual acuity, optical coherence tomography, electroretinogram and visual field testing. Extraocular findings included joint restriction and pains from an early age leading to bilateral hip replacement by age 30, aortic insufficiency, and hypertension. Genetic analysis was performed by whole genome sequencing filtered for a gene panel of 325 genes associated with retinal disease. Two compound heterozygous pathogenic variants were identified in GNPTG, c.347_349del and c.607dup. The diagnosis of mucolipidosis type III gamma was confirmed biochemically by measurement of increased activities of specific lysosomal enzymes in plasma. CONCLUSION: To our knowledge this is the first description of retinitis pigmentosa caused by compound heterozygous variants in GNPTG, providing further indications that late-onset retinal dystrophy is part of the phenotypic spectrum of mucolipidosis type III gamma.


Assuntos
Mucolipidoses , Distrofias Retinianas , Masculino , Humanos , Adulto , Pessoa de Meia-Idade , Mucolipidoses/diagnóstico , Mucolipidoses/genética , Sequenciamento Completo do Genoma , Eletrorretinografia , Transferases (Outros Grupos de Fosfato Substituídos)
10.
Nat Cell Biol ; 25(8): 1173-1184, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37488437

RESUMO

The migrasome is an organelle of migrating cells with diverse physiological functions. How migrasome formation is initiated is unknown. We found that sphingomyelin is enriched in migrasomes and identified sphingomyelin synthase 2 (SMS2) as an essential protein for migrasome biogenesis. SMS2 assembles into immobile foci that adhere on the basal membrane at the leading edge. When cells migrate away, the SMS2 foci 'move' out of cells and into retraction fibres, where they become migrasome formation sites and eventually grow into migrasomes. Mechanistically, SMS2 foci seed migrasomes by converting ceramide to sphingomyelin, which is essential for migrasome formation. Furthermore, CerS5, which is required for the synthesis of long-chain ceramide, and CERT, which transports ceramide from the endoplasmic reticulum to Golgi, are both required for migrasome formation. Our data reveal the essential role of ceramide and sphingomyelin in migrasome formation and suggest that SMS2 forms basal membrane-surface-connecting structures that pre-determine where migrasomes will grow.


Assuntos
Esfingomielinas , Transferases (Outros Grupos de Fosfato Substituídos) , Esfingomielinas/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Ceramidas/metabolismo , Retículo Endoplasmático/metabolismo
11.
Arterioscler Thromb Vasc Biol ; 43(7): 1251-1261, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37128925

RESUMO

BACKGROUND: Sphingomyelin (SM) and cholesterol are 2 key lipid partners on cell membranes and on lipoproteins. Many studies have indicated the influence of cholesterol on SM metabolism. This study examined the influence of SM biosynthesis on cholesterol metabolism. METHODS: Inducible global Sms1 KO (knockout)/global Sms2 KO mice were prepared to evaluate the effect of whole-body SM biosynthesis deficiency on lipoprotein metabolism. Tissue cholesterol, SM, ceramide, and glucosylceramide levels were measured. Triglyceride production rate and LDL (low-density lipoprotein) catabolism were measured. Lipid rafts were isolated and LDL receptor mass and function were evaluated. Also, the effects of exogenous sphingolipids on hepatocytes were investigated. RESULTS: We found that total SMS (SM synthase) depletion significantly reduced plasma SM levels. Also, the total deficiency significantly induced plasma cholesterol, apoB (apolipoprotein B), and apoE (apolipoprotein E) levels. Importantly, total SMS deficiency, but not SMS2 deficiency, dramatically decreased LDL receptors in the liver and attenuated LDL uptake through the receptor. Further, we found that total SMS deficiency greatly reduced LDL receptors in the lipid rafts, which contained significantly lower SM and significantly higher glucosylceramide, as well as cholesterol. Furthermore, we treated primary hepatocytes and Huh7 cells (a human hepatoma cell line) with SM, ceramide, or glucosylceramide, and we found that only SM could upregulate LDL receptor levels in a dose-dependent fashion. CONCLUSIONS: Whole-body SM biosynthesis plays an important role in LDL cholesterol catabolism. The total SMS deficiency, but not SMS2 deficiency, reduces LDL uptake and causes LDL cholesterol accumulation in the circulation. Given the fact that serum SM level is a risk factor for cardiovascular diseases, inhibiting SMS2 but not SMS1 should be the desirable approach.


Assuntos
Glucosilceramidas , Esfingomielinas , Camundongos , Humanos , Animais , LDL-Colesterol , Ceramidas/metabolismo , Colesterol/metabolismo , Receptores de LDL , Apolipoproteínas , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo
12.
Methods Mol Biol ; 2670: 145-163, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37184703

RESUMO

4'-Phosphopantetheinyl transferases (PPTases) play an essential role in activating the carrier protein domains of mega-synthases involved in primary and secondary metabolism and have been validated as promising drug targets in multiple pathogens. Monitoring phosphopantetheinylation of the non-ribosomal peptidase synthetase BpsA, which produces blue indigoidine pigment upon activation, is a useful strategy to screen chemical collections for inhibitors of a target PPTase. However, PPTases can exhibit carrier protein specificity and some medically important PPTases do not activate BpsA. Here, we describe how to conduct a directed evolution campaign to evolve the BpsA carrier protein domain for improved recognition by a candidate PPTase, as exemplified for the human Sfp-like PPTase. This method can be applied to other non-cognate PPTases for discovery of new drug candidates or chemical probes, or to enable development of next-generation biosensors that utilize BpsA as a reporter.


Assuntos
Proteínas de Transporte , Transferases , Humanos , Proteínas de Transporte/metabolismo , Transferases/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Proteínas de Bactérias/metabolismo
13.
PLoS Genet ; 19(5): e1010760, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-37200393

RESUMO

Heterozygous variants in the glucocerebrosidase (GBA) gene are common and potent risk factors for Parkinson's disease (PD). GBA also causes the autosomal recessive lysosomal storage disorder (LSD), Gaucher disease, and emerging evidence from human genetics implicates many other LSD genes in PD susceptibility. We have systemically tested 86 conserved fly homologs of 37 human LSD genes for requirements in the aging adult Drosophila brain and for potential genetic interactions with neurodegeneration caused by α-synuclein (αSyn), which forms Lewy body pathology in PD. Our screen identifies 15 genetic enhancers of αSyn-induced progressive locomotor dysfunction, including knockdown of fly homologs of GBA and other LSD genes with independent support as PD susceptibility factors from human genetics (SCARB2, SMPD1, CTSD, GNPTAB, SLC17A5). For several genes, results from multiple alleles suggest dose-sensitivity and context-dependent pleiotropy in the presence or absence of αSyn. Homologs of two genes causing cholesterol storage disorders, Npc1a / NPC1 and Lip4 / LIPA, were independently confirmed as loss-of-function enhancers of αSyn-induced retinal degeneration. The enzymes encoded by several modifier genes are upregulated in αSyn transgenic flies, based on unbiased proteomics, revealing a possible, albeit ineffective, compensatory response. Overall, our results reinforce the important role of lysosomal genes in brain health and PD pathogenesis, and implicate several metabolic pathways, including cholesterol homeostasis, in αSyn-mediated neurotoxicity.


Assuntos
Doença de Parkinson , alfa-Sinucleína , Animais , Humanos , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Animais Geneticamente Modificados , Drosophila/genética , Drosophila/metabolismo , Glucosilceramidase/genética , Glucosilceramidase/metabolismo , Lisossomos/metabolismo , Doença de Parkinson/patologia , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Envelhecimento/metabolismo
14.
J Exp Clin Cancer Res ; 42(1): 33, 2023 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-36691044

RESUMO

BACKGROUND: With increasing incidence and mortality, colorectal cancer (CRC) seriously endangers human health. LARP6, a member of La-related protein (LARP) family, is a RNA binding protein and probably associates with CRC progression, but its specific roles and mechanisms in CRC still remain unknown. METHOD: Quantitative real-time PCR (qPCR), western blot, and immunohistochemistry were employed to examine LARP6 expression in CRC tissues. Using the stable LARP6 overexpression or interference CRC cell lines, the effect of LARP6 on CRC progression were evaluated. High-throughput RNA immunoprecipitation sequencing (RIP-seq) and a series of relevant experiments were conducted to explain how LARP6 functions. SPSS software was used for statistical analysis. RESULT: In this study, we found that LARP6 expression is downregulated in CRC and correlates with patients' overall survival and relapse-free survival. Furthermore, altered LARP6 expression influences CRC cells invasion and metastasis. Mechanically, we discovered that LARP6 bind ZNF267 mRNA and regulated its stability and translation. LARP6 inhibited expression of SGMS2, a downstream target of ZNF267, resulting in ceramide and sphingomyelin imbalance in CRC cells. Interestingly, LARP6 also enhances autophagy activity of CRC cells, and the effect was at least partially determined by the inhibition of SGMS2-mediated sphingomyelin synthesis. CONCLUSION: Our study showed how LARP6/ZNF267/SGMS2 axis influence CRC progression, which contributes to further understanding of the molecular mechanisms underlying CRC development.


Assuntos
Neoplasias Colorretais , MicroRNAs , Proteínas Repressoras , Ribonucleoproteínas , Transferases (Outros Grupos de Fosfato Substituídos) , Humanos , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Colorretais/patologia , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Esfingomielinas , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas Repressoras/metabolismo
15.
J Cardiovasc Transl Res ; 16(1): 233-243, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-35972719

RESUMO

Nε-carboxymethyl-lysine (CML) is closely associated with vascular calcification in diabetes. Osteoclasts are the only cells with bone resorption activity that have the potential to reverse calcification. This study aimed to investigate the mechanism of CML in the bone resorption activity of macrophage-derived osteoclasts in diabetic calcified plaques. Macrophage-derived osteoclasts were found to be present in calcified plaques of the anterior tibial artery in patients with diabetic amputation. Furthermore, in vitro studies showed that CML induced the differentiation of macrophages into osteoclasts, although, the bone resorption activity of these macrophage-derived osteoclasts was impaired. CML significantly increased the levels of NFATc1and GNPTAB. In vivo studies showed that there was more calcium deposition and less TRAP was less in the CML group while this effect was reversed after silencing of NFATc1. In conclusion, CML mediates NFATc1-GNPTAB to regulate bone resorption activity of osteoclasts in diabetic calcified plaques. CML promotes macrophage differentiation into osteoclasts, but their function is impaired in diabetic calcified plaques through NFATc1-GNPTAB, which eventually leads to the further progression of vascular calcification in diabetes.


Assuntos
Reabsorção Óssea , Diabetes Mellitus , Placa Aterosclerótica , Calcificação Vascular , Humanos , Osteoclastos , Fatores de Transcrição , Diferenciação Celular , Fatores de Transcrição NFATC , Transferases (Outros Grupos de Fosfato Substituídos)
16.
Chem Phys Lipids ; 249: 105255, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36279928

RESUMO

Sphingomyelin synthase (SMS) synthesizes sphingomyelin (SM) from ceramide (Cer), a precursor of Cer. The effects of SMS deficiency on stratum corneum (SC) barrier function and SC lamellar structure are unknown. In this report, permeation of hydrophilic and lipophilic compounds through full-thickness skin or stripped skin of SMS2-knockout (KO) and wild-type (WT) mice was examined. Furthermore, small-angle and wide-angle X-ray scattering (SAXS and WAXS) measurements of the SC were performed as a function of temperature to analyze the lamellar structure and hydrocarbon chain packing, where a SC sample was changed from 10 °C to 120 °C at 2 °C/min and the X-ray diffraction profile in the small-angle region and the wide-angle region was observed. Skin permeability of the hydrophilic compound increased significantly for SMS2-KO mice when compared with that of WT mice. In contrast, no difference was observed in the penetration of lipophilic compounds in the skin of both SMS2-KO and WT mice. In SC of SMS2-KO mice, two sharp SAXS peaks were observed due to the lamellar structure with a repetition period of 4.8 nm. The WAXS revealed that the intensity ratio R0.42/0.37 of the 0.42 nm peak at 2.4 nm-1 to the 0.37 nm peak at 2.7 nm-1 was smaller in the SMS2-KO mouse than in the WT mouse. Due to the temperature dependence of the WAXS, the peaks of 2.4 and 2.7 nm-1 remained until the higher temperatures in SMS2-KO mouse SC than those in WT mouse SC. The results of X-ray diffraction suggest that deficiency of SMS2 may cause the appearance of highly ordered structures of SC, which in turn may reduce the barrier function of SC.


Assuntos
Epiderme , Transferases (Outros Grupos de Fosfato Substituídos) , Animais , Camundongos , Epiderme/anatomia & histologia , Epiderme/fisiopatologia , Camundongos Knockout , Espalhamento a Baixo Ângulo , Difração de Raios X , Transferases (Outros Grupos de Fosfato Substituídos)/deficiência , Transferases (Outros Grupos de Fosfato Substituídos)/genética
17.
Int J Mol Sci ; 23(18)2022 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-36142562

RESUMO

Ceramide levels controlled by the sphingomyelin (SM) cycle have essential roles in cancer cell fate through the regulation of cell proliferation, death, metastasis, and drug resistance. Recent studies suggest that exosomes confer cancer malignancy. However, the relationship between ceramide metabolism and exosome-mediated cancer malignancy is unclear. In this study, we elucidated the role of ceramide metabolism via the SM cycle in exosomes and drug resistance in human leukemia HL-60 and adriamycin-resistant HL-60/ADR cells. HL-60/ADR cells showed significantly increased exosome production and release compared with parental chemosensitive HL-60 cells. In HL-60/ADR cells, increased SM synthase (SMS) activity reduced ceramide levels, although released exosomes exhibited a high ceramide ratio in both HL-60- and HL-60/ADR-derived exosomes. Overexpression of SMS2 but not SMS1 suppressed intracellular ceramide levels and accelerated exosome production and release in HL-60 cells. Notably, HL-60/ADR exosomes conferred cell proliferation and doxorubicin resistance properties to HL-60 cells. Finally, microRNA analysis in HL-60 and HL-60/ADR cells and exosomes showed that miR-484 elevation in HL-60/ADR cells and exosomes was associated with exosome-mediated cell proliferation. This suggests that intracellular ceramide metabolism by SMS2 regulates exosome production and release, leading to acquisition of drug resistance and enhanced cell proliferation in leukemia cells.


Assuntos
Exossomos , Leucemia , MicroRNAs , Ceramidas/metabolismo , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Exossomos/metabolismo , Humanos , MicroRNAs/genética , Esfingomielinas/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)
18.
Science ; 378(6615): eabn5637, 2022 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-36074822

RESUMO

Mammalian cells can generate amino acids through macropinocytosis and lysosomal breakdown of extracellular proteins, which is exploited by cancer cells to grow in nutrient-poor tumors. Through genetic screens in defined nutrient conditions, we characterized LYSET, a transmembrane protein (TMEM251) selectively required when cells consume extracellular proteins. LYSET was found to associate in the Golgi with GlcNAc-1-phosphotransferase, which targets catabolic enzymes to lysosomes through mannose-6-phosphate modification. Without LYSET, GlcNAc-1-phosphotransferase was unstable because of a hydrophilic transmembrane domain. Consequently, LYSET-deficient cells were depleted of lysosomal enzymes and impaired in turnover of macropinocytic and autophagic cargoes. Thus, LYSET represents a core component of the lysosomal enzyme trafficking pathway, underlies the pathomechanism for hereditary lysosomal storage disorders, and may represent a target to suppress metabolic adaptations in cancer.


Assuntos
Complexo de Golgi , Doenças por Armazenamento dos Lisossomos , Lisossomos , Proteínas , Animais , Complexo de Golgi/metabolismo , Humanos , Doenças por Armazenamento dos Lisossomos/genética , Doenças por Armazenamento dos Lisossomos/metabolismo , Lisossomos/metabolismo , Camundongos , Transporte Proteico , Proteínas/genética , Proteínas/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo
19.
Science ; 378(6615): eabn5648, 2022 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-36074821

RESUMO

Lysosomes are key degradative compartments of the cell. Transport to lysosomes relies on GlcNAc-1-phosphotransferase-mediated tagging of soluble enzymes with mannose 6-phosphate (M6P). GlcNAc-1-phosphotransferase deficiency leads to the severe lysosomal storage disorder mucolipidosis II (MLII). Several viruses require lysosomal cathepsins to cleave structural proteins and thus depend on functional GlcNAc-1-phosphotransferase. We used genome-scale CRISPR screens to identify lysosomal enzyme trafficking factor (LYSET, also named TMEM251) as essential for infection by cathepsin-dependent viruses including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). LYSET deficiency resulted in global loss of M6P tagging and mislocalization of GlcNAc-1-phosphotransferase from the Golgi complex to lysosomes. Lyset knockout mice exhibited MLII-like phenotypes, and human pathogenic LYSET alleles failed to restore lysosomal sorting defects. Thus, LYSET is required for correct functioning of the M6P trafficking machinery and mutations in LYSET can explain the phenotype of the associated disorder.


Assuntos
COVID-19 , Lisossomos , Mucolipidoses , Proteínas , Animais , COVID-19/genética , Catepsinas/metabolismo , Humanos , Lisossomos/metabolismo , Manose/metabolismo , Camundongos , Camundongos Knockout , Mucolipidoses/genética , Mucolipidoses/metabolismo , Proteínas/genética , Transferases (Outros Grupos de Fosfato Substituídos)
20.
Nat Commun ; 13(1): 5351, 2022 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-36096887

RESUMO

The mannose-6-phosphate (M6P) biosynthetic pathway for lysosome biogenesis has been studied for decades and is considered a well-understood topic. However, whether this pathway is regulated remains an open question. In a genome-wide CRISPR/Cas9 knockout screen, we discover TMEM251 as the first regulator of the M6P modification. Deleting TMEM251 causes mistargeting of most lysosomal enzymes due to their loss of M6P modification and accumulation of numerous undigested materials. We further demonstrate that TMEM251 localizes to the Golgi and is required for the cleavage and activity of GNPT, the enzyme that catalyzes M6P modification. In zebrafish, TMEM251 deletion leads to severe developmental defects including heart edema and skeletal dysplasia, which phenocopies Mucolipidosis Type II. Our discovery provides a mechanism for the newly discovered human disease caused by TMEM251 mutations. We name TMEM251 as GNPTAB cleavage and activity factor (GCAF) and its related disease as Mucolipidosis Type V.


Assuntos
Proteínas de Membrana , Mucolipidoses , Peixe-Zebra , Animais , Humanos , Lisossomos/metabolismo , Manosefosfatos/metabolismo , Proteínas de Membrana/metabolismo , Mucolipidoses/genética , Mucolipidoses/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Peixe-Zebra/metabolismo
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