RESUMO
The lack of effective vaccines and the development of resistance to the current treatments highlight the urgent need for new anti-leishmanials. Sphingolipid metabolism has been proposed as a promising source of Leishmania-specific targets as these lipids are key structural components of the eukaryotic plasma membrane and are involved in distinct cellular events. Inositol phosphorylceramide (IPC) is the primary sphingolipid in the Leishmania species and is the product of a reaction mediated by IPC synthase (IPCS). The antihistamine clemastine fumarate has been identified as an inhibitor of IPCS in L. major and a potent anti-leishmanial in vivo. Here we sought to further examine the target of this compound in the more tractable species L. mexicana, using an approach combining genomic, proteomic, metabolomic and lipidomic technologies, with molecular and biochemical studies. While the data demonstrated that the response to clemastine fumarate was largely conserved, unexpected disturbances beyond sphingolipid metabolism were identified. Furthermore, while deletion of the gene encoding LmxIPCS had little impact in vitro, it did influence clemastine fumarate efficacy and, importantly, in vivo pathogenicity. Together, these data demonstrate that clemastine does inhibit LmxIPCS and cause associated metabolic disturbances, but its primary target may lie elsewhere.
Assuntos
Antiprotozoários , Antiprotozoários/farmacologia , Antiprotozoários/química , Esfingolipídeos/metabolismo , Hexosiltransferases/genética , Hexosiltransferases/metabolismo , Hexosiltransferases/antagonistas & inibidores , Leishmania/efeitos dos fármacos , Leishmania/genética , Leishmania/enzimologia , Animais , Leishmania mexicana/efeitos dos fármacos , Leishmania mexicana/genética , Leishmania mexicana/enzimologia , Glicoesfingolipídeos/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismoRESUMO
Ceramide (Cer) is synthesized de novo in the bilayer of the endoplasmic reticulum and transported to the cytosolic leaflet of the trans-Golgi apparatus for sphingomyelin (SM) synthesis. As the active site of SM synthase (SMS) is located on the luminal side of the Golgi membrane, Cer translocates to the lumen via transbilayer movement for SM synthesis. However, the mechanism of transbilayer movement is not fully understood. As the Cer-related translocases seem to localize near the SMS, the protein was identified using proximity-dependent biotin identification proteomics. Phospholipid scramblase 1 (PLSCR1), which is thought to act as a scramblase for phosphatidylserine and phosphatidylethanolamine, was identified as a protein proximal to the SMS isoforms SMS1 and SMS2. Although five isoforms of PLSCR have been reported in humans, only PLSCR1, PLSCR3, and PLSCR4 are expressed in HEK293T cells. Confocal microscopic analysis showed that PLSCR1 and PLSCR4 partially co-localized with p230, a trans-Golgi network marker, where SMS isoforms are localized. We established CRISPR/Cas9-mediated PLSCR1, PLSCR3, and PLSCR4 single-knockout cells and PLSCR1, 3, 4 triple knockout HEK293T cells. Liquid chromatography-tandem mass spectrometry revealed that the levels of species with distinct acyl chains in Cer and SM were not significantly different in single knockout cells or in the triple knockout cells compared to the wild-type cells. Our findings suggest that PLSCR1 is localized in the vicinity of SMS isoforms, however is not involved in the transbilayer movement of Cer for SM synthesis.
Assuntos
Proteínas de Transferência de Fosfolipídeos , Esfingomielinas , Transferases (Outros Grupos de Fosfato Substituídos) , Humanos , Proteínas de Transferência de Fosfolipídeos/metabolismo , Proteínas de Transferência de Fosfolipídeos/genética , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Células HEK293 , Esfingomielinas/metabolismo , Esfingomielinas/biossíntese , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Isoenzimas/metabolismo , Isoenzimas/genética , Complexo de Golgi/metabolismo , Complexo de Golgi/enzimologiaRESUMO
Renal ischemia/reperfusion is a serious condition that not only causes acute kidney injury, a severe clinical syndrome with high mortality, but is also an inevitable part of kidney transplantation or other kidney surgeries. Alterations of oxygen levels during ischemia/reperfusion, namely hypoxia/reoxygenation, disrupt mitochondrial metabolism and induce structural changes that lead to cell death. A signature mitochondrial phospholipid, cardiolipin, with many vital roles in mitochondrial homeostasis, is one of the key players in hypoxia/reoxygenation-induced mitochondrial damage. In this study, we analyze the effect of hypoxia/reoxygenation on human renal proximal tubule epithelial cell (RPTEC) cardiolipins, as well as their metabolism and mitochondrial functions. RPTEC cells were placed in a hypoxic chamber with a 2% oxygen atmosphere for 24 h to induce hypoxia; then, they were replaced back into regular growth conditions for 24 h of reoxygenation. Surprisingly, after 24 h, hypoxia cardiolipin levels substantially increased and remained higher than control levels after 24 h of reoxygenation. This was explained by significantly elevated levels of cardiolipin synthase and lysocardiolipin acyltransferase 1 (LCLAT1) gene expression and protein levels. Meanwhile, hypoxia/reoxygenation decreased ADP-dependent mitochondrial respiration rates and oxidative phosphorylation capacity and increased reactive oxygen species generation. Our findings suggest that hypoxia/reoxygenation induces cardiolipin remodeling in response to reduced mitochondrial oxidative phosphorylation in a way that protects mitochondrial function.
Assuntos
Cardiolipinas , Hipóxia Celular , Mitocôndrias , Oxigênio , Espécies Reativas de Oxigênio , Humanos , Cardiolipinas/metabolismo , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Oxigênio/metabolismo , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , Túbulos Renais Proximais/citologia , Fosforilação Oxidativa , Rim/metabolismo , Rim/patologia , Linhagem Celular , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Proteínas de MembranaRESUMO
Sphingomyelin synthase (SMS) is a sphingolipid-metabolizing enzyme involved in the de novo synthesis of sphingomyelin (SM) from ceramide (Cer). Recent studies have indicated that SMS is a key therapeutic target for metabolic diseases such as fatty liver, type 2 diabetes, atherosclerosis, and colorectal cancer. However, very few SMS inhibitors have been identified because of the limited sensitivity and selectivity of the current fluorescence-based screening assay. In this study, we developed a simple cell-based assay coupled with liquid chromatography/tandem mass spectrometry (LC-MS/MS) to screen for SMS inhibitors. HeLa cells stably expressing SMS1 or SMS2 were used for the screening. A non-fluorescent unnatural C6-Cer was used as a substrate for SMS to produce C6-SM. C6-Cer and C6-SM levels in the cells were monitored and quantified using LC-MS/MS. The activity of ginkgolic acid C15:1 (GA), a known SMS inhibitor, was measured. GA had half-maximal inhibitory concentrations of 5.5 µM and 3.6 µM for SMS1 and SMS2, respectively. To validate these findings, hSMS1 and hSMS2 proteins were optimized for molecular docking studies. In silico analyses were conducted to assess the interaction of GA with SMS1 and SMS2, and its binding affinity. This study offers an analytical approach for screening novel SMS inhibitors and provides in silico support for the experimental findings.
Assuntos
Espectrometria de Massas em Tandem , Transferases (Outros Grupos de Fosfato Substituídos) , Humanos , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/antagonistas & inibidores , Células HeLa , Espectrometria de Massas em Tandem/métodos , Cromatografia Líquida/métodos , Simulação de Acoplamento Molecular , Inibidores Enzimáticos/farmacologia , Proteínas do Tecido Nervoso/metabolismo , Proteínas de MembranaRESUMO
The membrane raft accommodates the key enzymes synthesizing amyloid ß (Aß). One of the two characteristic components of the membrane raft, cholesterol, is well known to promote the key enzymes that produce amyloid-ß (Aß) and exacerbate Alzheimer's disease (AD) pathogenesis. Given that the raft is a physicochemical platform for the sound functioning of embedded bioactive proteins, the other major lipid component sphingomyelin may also be involved in AD. Here we knocked out the sphingomyelin synthase 2 gene (SMS2) in 3xTg AD model mice by hybridization, yielding SMS2KO mice (4S mice). The novel object recognition test in 9/10-month-old 4S mice showed that cognitive impairment in 3xTg mice was alleviated by SMS2KO, though performance in the Morris water maze (MWM) was not improved. The tail suspension test detected a depressive trait in 4S mice, which may have hindered the manifestation of performance in the wet, stressful environment of MWM. In the hippocampal CA1, hyperexcitability in 3xTg was also found alleviated by SMS2KO. In the hippocampal dentate gyrus of 4S mice, the number of neurons positive with intracellular Aß or its precursor proteins, the hallmark of young 3xTg mice, is reduced to one-third, suggesting an SMS2KO-led suppression of syntheses of those peptides in the dentate gyrus. Although we previously reported that large-conductance calcium-activated potassium (BK) channels are suppressed in 3xTg mice and their recovery relates to cognitive amelioration, no changes occurred by hybridization. Sphingomyelin in the membrane raft may serve as a novel target for AD drugs.
Assuntos
Doença de Alzheimer , Disfunção Cognitiva , Modelos Animais de Doenças , Camundongos Knockout , Camundongos Transgênicos , Transferases (Outros Grupos de Fosfato Substituídos) , Animais , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Disfunção Cognitiva/metabolismo , Disfunção Cognitiva/genética , Camundongos , Peptídeos beta-Amiloides/metabolismo , Masculino , Aprendizagem em Labirinto/fisiologia , Hipocampo/metabolismo , Camundongos Endogâmicos C57BLRESUMO
Glycerophospholipids have emerged as a significant contributor to the intracellular growth of pathogenic protist Toxoplasma gondii. Phosphatidylserine (PtdSer) is one such lipid, attributed to the locomotion and motility-dependent invasion and egress events in its acutely infectious tachyzoite stage. However, the de novo synthesis of PtdSer and the importance of the pathway in tachyzoites remain poorly understood. We show that a base-exchange-type PtdSer synthase (PSS) located in the parasite's endoplasmic reticulum produces PtdSer, which is rapidly converted to phosphatidylethanolamine (PtdEtn) by PtdSer decarboxylase (PSD) activity. The PSS-PSD pathway enables the synthesis of several lipid species, including PtdSer (16:0/18:1) and PtdEtn (18:2/20:4, 18:1/18:2 and 18:2/22:5). The PSS-depleted strain exhibited a lower abundance of the major ester-linked PtdEtn species and concurrent accrual of host-derived ether-PtdEtn species. Most phosphatidylthreonine (PtdThr) species-an exclusive natural analog of PtdSer, also made in the endoplasmic reticulum-were repressed. PtdSer species, however, remained largely unaltered, likely due to the serine-exchange reaction of PtdThr synthase in favor of PtdSer upon PSS depletion. Not least, the loss of PSS abrogated the lytic cycle of tachyzoites, impairing the cell division, motility, and egress. In a nutshell, our data demonstrate a critical role of PSS in the biogenesis of PtdSer and PtdEtn species and its physiologically essential repurposing for the asexual reproduction of a clinically relevant intracellular pathogen.
Assuntos
Retículo Endoplasmático , Toxoplasma , Toxoplasma/enzimologia , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/enzimologia , Humanos , Fosfatidilserinas/metabolismo , CDPdiacilglicerol-Serina O-Fosfatidiltransferase/metabolismo , CDPdiacilglicerol-Serina O-Fosfatidiltransferase/genética , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/genética , CarboxiliasesRESUMO
Polycistronic transcription and translation of ymdB-clsC have been thought to be required for full activity of ClsC. The authentic initiation codon of the clsC gene is present within the open reading frame of the upstream located ymdB gene. ClsC translated from authentic initiation codon drives cardiolipin (CL) synthesis without transcriptionally paired YmdB. YmdB is not necessary for the substrate specificity of ClsC utilizing phosphatidylethanolamine (PE) as a co-substrate.
Assuntos
Cardiolipinas , Proteínas de Escherichia coli , Transferases (Outros Grupos de Fosfato Substituídos) , Cardiolipinas/metabolismo , Cardiolipinas/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Fosfatidiletanolaminas/metabolismo , Especificidade por Substrato , Transcrição Gênica , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismoRESUMO
Human placenta is an intensively growing tissue. Phosphatidylinositol (PI) and its derivatives are part of the signaling pathway in the regulation of trophoblast cell differentiation. There are two different enzymes that take part in the direct PI synthesis: phosphatidylinositol synthase (PIS) and inositol exchange enzyme (IE). The presence of PIS is known in the human placenta, but IE activity has not been documented before. In our study, we describe the physiological properties of the two enzymes in vitro. PIS and IE were studied in different Mn2+ and Mg2+ concentrations that enabled us to separate the individual enzyme activities. Enzyme activity was measured by incorporation of 3[H]inositol in human primordial placenta tissue or microsomes. Optimal PIS activity was achieved between 0.5 and 2.0 mM Mn2+ concentration, but higher concentrations inhibit enzyme activity. In the presence of Mg2+, the enzyme activity increases continuously up to a concentration of 100 mM. PIS was inhibited by nucleoside di- and tri-phosphates. PI production increases between 0.1 and 10 mM Mn2+ concentration. The incorporation of [3H]inositol into PI increased by 57% when adding stabile GTP analog. The described novel pathway of inositol synthesis may provide an additional therapeutic approach of inositol supplementation before and during pregnancy.
Assuntos
Inositol , Fosfatidilinositóis , Feminino , Gravidez , Humanos , Inositol/farmacologia , Fosfatidilinositóis/metabolismo , CDP-Diacilglicerol-Inositol 3-Fosfatidiltransferase , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Placenta/metabolismoRESUMO
In a vast majority of bacteria, protozoa and plants, the methylerythritol phosphate (MEP) pathway is utilized for the synthesis of isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP), which are precursors for isoprenoids. Isoprenoids, such as cholesterol and coenzyme Q, play a variety of crucial roles in physiological activities, including cell-membrane formation, protein degradation, cell apoptosis, and transcription regulation. In contrast, humans employ the mevalonate (MVA) pathway for the production of IDP and DMADP, rendering proteins in the MEP pathway appealing targets for antimicrobial agents. This pathway consists of seven consecutive enzymatic reactions, of which 4-diphosphocytidyl-2C-methyl-D-erythritol synthase (IspD) and 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF) catalyze the third and fifth steps, respectively. In this study, we characterized the enzymatic activities and protein structures of Helicobacter pylori IspDF and Acinetobacter baumannii IspD. Then, using the direct interaction-based thermal shift assay, we conducted a compound screening of an approved drug library and identified 27 hit compounds potentially binding to AbIspD. Among them, two natural products, rosmarinic acid and tanshinone IIA sodium sulfonate, exhibited inhibitory activities against HpIspDF and AbIspD, by competing with one of the substrates, MEP. Moreover, tanshinone IIA sodium sulfonate also demonstrated certain antibacterial effects against H. pylori. In summary, we identified two IspD inhibitors from approved ingredients, broadening the scope for antibiotic discovery targeting the MEP pathway.
Assuntos
Acinetobacter baumannii , Antibacterianos , Helicobacter pylori , Hemiterpenos , Helicobacter pylori/efeitos dos fármacos , Helicobacter pylori/enzimologia , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/enzimologia , Antibacterianos/farmacologia , Inibidores Enzimáticos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Produtos Biológicos/farmacologia , Produtos Biológicos/química , Compostos Organofosforados/farmacologia , Humanos , Transferases (Outros Grupos de Fosfato Substituídos)/antagonistas & inibidores , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismoRESUMO
Sphingomyelin (SM) has key roles in modulating mammalian membrane properties and serves as an important pool for bioactive molecules. SM biosynthesis is mediated by the sphingomyelin synthase (SMS) family, comprising SMS1, SMS2 and SMS-related (SMSr) members. Although SMS1 and SMS2 exhibit SMS activity, SMSr possesses ceramide phosphoethanolamine synthase activity. Here we determined the cryo-electron microscopic structures of human SMSr in complexes with ceramide, diacylglycerol/phosphoethanolamine and ceramide/phosphoethanolamine (CPE). The structures revealed a hexameric arrangement with a reaction chamber located between the transmembrane helices. Within this structure, a catalytic pentad E-H/D-H-D was identified, situated at the interface between the lipophilic and hydrophilic segments of the reaction chamber. Additionally, the study unveiled the two-step synthesis process catalyzed by SMSr, involving PE-PLC (phosphatidylethanolamine-phospholipase C) hydrolysis and the subsequent transfer of the phosphoethanolamine moiety to ceramide. This research provides insights into the catalytic mechanism of SMSr and expands our understanding of sphingolipid metabolism.
Assuntos
Microscopia Crioeletrônica , Modelos Moleculares , Esfingomielinas , Transferases (Outros Grupos de Fosfato Substituídos) , Humanos , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/química , Esfingomielinas/metabolismo , Esfingomielinas/química , Esfingomielinas/biossíntese , Ceramidas/metabolismo , Ceramidas/química , Etanolaminas/metabolismo , Etanolaminas/química , Fosfatidiletanolaminas/metabolismo , Fosfatidiletanolaminas/química , Diglicerídeos/metabolismo , Diglicerídeos/química , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas de MembranaRESUMO
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)/metabolismoRESUMO
Sphingomyelin synthase (SMS)-related protein (SMSr) is a phosphatidylethanolamine phospholipase C (PE-PLC) that is conserved and ubiquitous in mammals. However, its biological function is still not clear. We previously observed that SMS1 deficiency-mediated glucosylceramide accumulation caused nonalcoholic fatty liver diseases (NAFLD), including nonalcoholic steatohepatitis (NASH) and liver fibrosis. Here, first, we evaluated high-fat diet/fructose-induced NAFLD in Smsr KO and WT mice. Second, we evaluated whether SMSr deficiency can reverse SMS1 deficiency-mediated NAFLD, using Sms1/Sms2 double and Sms1/Sms2/Smsr triple KO mice. We found that SMSr/PE-PLC deficiency attenuated high-fat diet/fructose-induced fatty liver and NASH, and attenuated glucosylceramide accumulation-induced NASH, fibrosis, and tumor formation. Further, we found that SMSr/PE-PLC deficiency reduced the expression of many inflammatory cytokines and fibrosis-related factors, and PE supplementation in vitro or in vivo mimicked the condition of SMSr/PE-PLC deficiency. Furthermore, we demonstrated that SMSr/PE-PLC deficiency or PE supplementation effectively prevented membrane-bound ß-catenin transfer to the nucleus, thereby preventing tumor-related gene expression. Finally, we observed that patients with NASH had higher SMSr protein levels in the liver, lower plasma PE levels, and lower plasma PE/phosphatidylcholine ratios, and that human plasma PE levels are negatively associated with tumor necrosis factor-α and transforming growth factor ß1 levels. In conclusion, SMSr/PE-PLC deficiency causes PE accumulation, which can attenuate fatty liver, NASH, and fibrosis. These results suggest that SMSr/PE-PLC inhibition therapy may mitigate NAFLD.
Assuntos
Neoplasias , Hepatopatia Gordurosa não Alcoólica , Transferases (Outros Grupos de Fosfato Substituídos) , Animais , Humanos , Camundongos , Frutose/efeitos adversos , Glucosilceramidas/metabolismo , Fígado/metabolismo , Cirrose Hepática/patologia , Neoplasias/genética , Neoplasias/metabolismo , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/metabolismo , Fosfatidiletanolaminas/sangue , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Camundongos Knockout , Masculino , Feminino , Dieta Hiperlipídica/efeitos adversosRESUMO
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/metabolismoRESUMO
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/metabolismoRESUMO
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/metabolismoRESUMO
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)/metabolismoRESUMO
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 , Antígeno SS-BRESUMO
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/metabolismoRESUMO
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)/metabolismoRESUMO
The sphingomyelin synthase (SMS) gene family has three members: SMS1 and SMS2 have SM synthase activity, while SMS-related protein (SMSr) has no SM synthase activity but has ceramide phosphorylethanolamine (CPE) synthase activity in vitro. Recently, we found that SMS family members are a group of phospholipase Cs (PLC). SMS1 and SMS2 are two phosphatidylcholine (PC)-PLCs and SMSr is a phosphatidylethanolamine (PE)-PLC. SMS family members not only influence SM levels but also influence the levels of diacylglycerol (DAG), PC, PE, and glycosphingolipids, thus influencing cell functions. In this chapter, we will discuss the recent progress in the research field of SMS family and will focus on its impact on metabolic diseases.