RESUMO
Metabolically healthy obesity refers to obese individuals who do not develop metabolic disorders. These people store fat in subcutaneous adipose tissue (SAT) rather than in visceral adipose tissue (VAT). However, the molecules participating in this specific scenario remain elusive. Rab18, a lipid droplet (LD)-associated protein, mediates the contact between the endoplasmic reticulum (ER) and LDs to facilitate LD growth and maturation. In the present study, we show that the protein level of Rab18 is specifically upregulated in the SAT of obese people and mice. Rab18 adipocyte-specific knockout (Rab18 AKO) mice had a decreased volume ratio of SAT to VAT compared with wildtype mice. When subjected to high-fat diet (HFD), Rab18 AKO mice had increased ER stress and inflammation, reduced adiponectin, and decreased triacylglycerol (TAG) accumulation in SAT. In contrast, TAG accumulation in VAT, brown adipose tissue (BAT) or liver of Rab18 AKO mice had a moderate increase without ER stress stimulation. Rab18 AKO mice developed insulin resistance and systematic inflammation. Rab18 AKO mice maintained body temperature in response to acute and chronic cold induction with a thermogenic SAT, similar to the counterpart mice. Furthermore, Rab18-deficient 3T3-L1 adipocytes were more prone to palmitate-induced ER stress, indicating the involvement of Rab18 in alleviating lipid toxicity. Rab18 AKO mice provide a good animal model to investigate metabolic disorders such as impaired SAT. In conclusion, our studies reveal that Rab18 is a key and specific regulator that maintains the proper functions of SAT by alleviating lipid-induced ER stress.
Assuntos
Dieta Hiperlipídica , Estresse do Retículo Endoplasmático , Homeostase , Camundongos Knockout , Obesidade , Gordura Subcutânea , Proteínas rab de Ligação ao GTP , Animais , Obesidade/metabolismo , Obesidade/genética , Obesidade/etiologia , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab de Ligação ao GTP/genética , Camundongos , Gordura Subcutânea/metabolismo , Humanos , Masculino , Dieta Hiperlipídica/efeitos adversos , Doenças Metabólicas/metabolismo , Doenças Metabólicas/etiologia , Doenças Metabólicas/prevenção & controle , Doenças Metabólicas/genética , Adipócitos/metabolismo , Resistência à Insulina , Células 3T3-L1 , Camundongos Endogâmicos C57BL , Triglicerídeos/metabolismo , Tecido Adiposo Marrom/metabolismo , Inflamação/metabolismo , Gotículas Lipídicas/metabolismo , Gordura Intra-Abdominal/metabolismo , FemininoRESUMO
Understanding protective immunity to COVID-19 facilitates preparedness for future pandemics and combats new SARS-CoV-2 variants emerging in the human population. Neutralizing antibodies have been widely studied; however, on the basis of large-scale exome sequencing of protected versus severely ill patients with COVID-19, local cell-autonomous defence is also crucial1-4. Here we identify phospholipid scramblase 1 (PLSCR1) as a potent cell-autonomous restriction factor against live SARS-CoV-2 infection in parallel genome-wide CRISPR-Cas9 screens of human lung epithelia and hepatocytes before and after stimulation with interferon-γ (IFNγ). IFNγ-induced PLSCR1 not only restricted SARS-CoV-2 USA-WA1/2020, but was also effective against the Delta B.1.617.2 and Omicron BA.1 lineages. Its robust activity extended to other highly pathogenic coronaviruses, was functionally conserved in bats and mice, and interfered with the uptake of SARS-CoV-2 in both the endocytic and the TMPRSS2-dependent fusion routes. Whole-cell 4Pi single-molecule switching nanoscopy together with bipartite nano-reporter assays found that PLSCR1 directly targeted SARS-CoV-2-containing vesicles to prevent spike-mediated fusion and viral escape. A PLSCR1 C-terminal ß-barrel domain-but not lipid scramblase activity-was essential for this fusogenic blockade. Our mechanistic studies, together with reports that COVID-associated PLSCR1 mutations are found in some susceptible people3,4, identify an anti-coronavirus protein that interferes at a late entry step before viral RNA is released into the host-cell cytosol.
Assuntos
COVID-19 , Proteínas de Transferência de Fosfolipídeos , SARS-CoV-2 , Animais , Humanos , Camundongos , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Quirópteros , COVID-19/imunologia , COVID-19/metabolismo , COVID-19/prevenção & controle , COVID-19/virologia , Sequenciamento do Exoma , Hepatócitos/imunologia , Hepatócitos/metabolismo , Interferon gama/imunologia , Pulmão/imunologia , Pulmão/metabolismo , Fusão de Membrana , Proteínas de Transferência de Fosfolipídeos/química , Proteínas de Transferência de Fosfolipídeos/genética , Proteínas de Transferência de Fosfolipídeos/imunologia , Proteínas de Transferência de Fosfolipídeos/metabolismo , SARS-CoV-2/classificação , SARS-CoV-2/imunologia , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidade , Internalização do VírusRESUMO
Somatic hypermutation (SHM) produces point mutations in immunoglobulin (Ig) genes in B cells when uracils created by the activation-induced deaminase are processed in a mutagenic manner by enzymes of the base excision repair (BER) and mismatch repair (MMR) pathways. Such uracil processing creates DNA strand breaks and is susceptible to the generation of deleterious deletions. Here, we demonstrate that the DNA repair factor HMCES strongly suppresses deletions without significantly affecting other parameters of SHM in mouse and human B cells, thereby facilitating the production of antigen-specific antibodies. The deletion-prone repair pathway suppressed by HMCES operates downstream from the uracil glycosylase UNG and is mediated by the combined action of BER factor APE2 and MMR factors MSH2, MSH6, and EXO1. HMCES's ability to shield against deletions during SHM requires its capacity to form covalent cross-links with abasic sites, in sharp contrast to its DNA end-joining role in class switch recombination but analogous to its genome-stabilizing role during DNA replication. Our findings lead to a novel model for the protection of Ig gene integrity during SHM in which abasic site cross-linking by HMCES intercedes at a critical juncture during processing of vulnerable gapped DNA intermediates by BER and MMR enzymes.
Assuntos
Genes de Imunoglobulinas , Hipermutação Somática de Imunoglobulina , Animais , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , DNA/genética , Proteínas de Ligação a DNA , Genes de Imunoglobulinas/genética , Switching de Imunoglobulina/genética , Camundongos , Hipermutação Somática de Imunoglobulina/genética , UracilaRESUMO
Activation of cell-autonomous defense by the immune cytokine interferon-γ (IFN-γ) is critical to the control of life-threatening infections in humans. IFN-γ induces the expression of hundreds of host proteins in all nucleated cells and tissues, yet many of these proteins remain uncharacterized. We screened 19,050 human genes by CRISPR-Cas9 mutagenesis and identified IFN-γ-induced apolipoprotein L3 (APOL3) as a potent bactericidal agent protecting multiple non-immune barrier cell types against infection. Canonical apolipoproteins typically solubilize mammalian lipids for extracellular transport; APOL3 instead targeted cytosol-invasive bacteria to dissolve their anionic membranes into human-bacterial lipoprotein nanodiscs detected by native mass spectrometry and visualized by single-particle cryo-electron microscopy. Thus, humans have harnessed the detergent-like properties of extracellular apolipoproteins to fashion an intracellular lysin, thereby endowing resident nonimmune cells with a mechanism to achieve sterilizing immunity.
Assuntos
Apolipoproteínas L/metabolismo , Membrana Celular/metabolismo , Citosol/microbiologia , Bactérias Gram-Negativas/fisiologia , Interferon gama/imunologia , Apolipoproteínas L/química , Apolipoproteínas L/genética , Membrana Externa Bacteriana/metabolismo , Bacteriólise , Sistemas CRISPR-Cas , Membrana Celular/química , Membrana Celular/ultraestrutura , Permeabilidade da Membrana Celular , Células Cultivadas , Detergentes/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Edição de Genes , Bactérias Gram-Negativas/imunologia , Bactérias Gram-Negativas/patogenicidade , Bactérias Gram-Negativas/ultraestrutura , Humanos , Imunidade Inata , Lipoproteínas/química , Viabilidade Microbiana , Antígenos O/metabolismo , Domínios Proteicos , Salmonella typhimurium/imunologia , Salmonella typhimurium/patogenicidade , Salmonella typhimurium/fisiologia , Salmonella typhimurium/ultraestrutura , SolubilidadeRESUMO
Lipid droplets (LDs) are dynamic cellular organelles found in most eukaryotic cells. Lipid incorporation from endoplasmic reticulum (ER) to LD is important in controlling LD growth and intracellular lipid homeostasis. However, the molecular link that mediates ER and LD cross talk remains elusive. Here, we describe the methodology used to characterize the function of Rab18 in regulating LD homeostasis and LD-ER contact. First, we focus on the quantitative assay used to measure intracellular LDs morphological changes. This is followed by a detailed description of the use of the APEX-label technology in combination with electron microscope (EM) to visualize ER-LD contact sites. These assays are valuable for the investigation of LD-associated proteins such as Rab18 in establishing membrane contact sites between LDs and other subcellular organelles.
Assuntos
Retículo Endoplasmático , Retículo Endoplasmático/metabolismo , Gotículas Lipídicas/metabolismo , Metabolismo dos Lipídeos , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Obesity is characterized by excessive fatty acid conversion to triacylglycerols (TAGs) in adipose tissues. However, how signaling networks sense fatty acids and connect to the stimulation of lipid synthesis remains elusive. Here, we show that homozygous knock-in mice carrying a point mutation at the Ser86 phosphorylation site of acetyltransferase Tip60 (Tip60 SA/SA ) display remarkably reduced body fat mass, and Tip60 SA/SA females fail to nurture pups to adulthood due to severely reduced milk TAGs. Mechanistically, fatty acids stimulate Tip60-dependent acetylation and endoplasmic reticulum translocation of phosphatidic acid phosphatase lipin 1 to generate diacylglycerol for TAG synthesis, which is repressed by deacetylase Sirt1. Inhibition of Tip60 activity strongly blocks fatty acid-induced TAG synthesis while Sirt1 suppression leads to increased adiposity. Genetic analysis of loss-of-function mutants in Saccharomyces cerevisiae reveals a requirement of ESA1, yeast ortholog of Tip60, in TAG accumulation. These findings uncover a conserved mechanism linking fatty acid sensing to fat synthesis.
Assuntos
Retículo Endoplasmático/enzimologia , Lisina Acetiltransferase 5/metabolismo , Proteínas Nucleares/metabolismo , Fosfatidato Fosfatase/metabolismo , Transativadores/metabolismo , Triglicerídeos/biossíntese , Acetilação , Animais , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Ácidos Graxos/metabolismo , Feminino , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Cinética , Lisina Acetiltransferase 5/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Nucleares/genética , Fosfatidato Fosfatase/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Sirtuína 1/genética , Sirtuína 1/metabolismo , Transativadores/genética , Triglicerídeos/químicaRESUMO
Lipid incorporation from endoplasmic reticulum (ER) to lipid droplet (LD) is important in controlling LD growth and intracellular lipid homeostasis. However, the molecular link mediating ER and LD cross talk remains elusive. Here, we identified Rab18 as an important Rab guanosine triphosphatase in controlling LD growth and maturation. Rab18 deficiency resulted in a drastically reduced number of mature LDs and decreased lipid storage, and was accompanied by increased ER stress. Rab3GAP1/2, the GEF of Rab18, promoted LD growth by activating and targeting Rab18 to LDs. LD-associated Rab18 bound specifically to the ER-associated NAG-RINT1-ZW10 (NRZ) tethering complex and their associated SNAREs (Syntaxin18, Use1, BNIP1), resulting in the recruitment of ER to LD and the formation of direct ER-LD contact. Cells with defects in the NRZ/SNARE complex function showed reduced LD growth and lipid storage. Overall, our data reveal that the Rab18-NRZ-SNARE complex is critical protein machinery for tethering ER-LD and establishing ER-LD contact to promote LD growth.
Assuntos
Retículo Endoplasmático/metabolismo , Gotículas Lipídicas/metabolismo , Proteínas SNARE/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Células 3T3-L1 , Animais , Retículo Endoplasmático/genética , Camundongos , Proteínas SNARE/genética , Proteínas rab de Ligação ao GTP/genéticaRESUMO
Skeletal muscle absorbs long-chain fatty acids (LCFAs) that are either oxidized in mitochondria or temporarily stored as triglycerides in lipid droplets (LDs). So far, it is still not fully understood how lipid uptake and storage are regulated in muscle and whether these are important for whole-body lipid homeostasis. Here we show that the small GTPase Rab8a regulates lipid uptake and storage in skeletal muscle. Muscle-specific Rab8a deletion caused hyperlipidemia and exacerbated hepatosteatosis induced by a high-fat diet. Mechanistically, Rab8a deficiency decreased LCFA entry into skeletal muscle and inhibited LD fusion in muscle cells. Consequently, blood lipid levels were elevated and stimulated hepatic mammalian target of rapamycin, which enhanced hepatosteatosis by upregulating hepatic lipogenesis and cholesterol biosynthesis. Our results demonstrate the significance of lipid uptake and storage in muscle in regulating whole-body lipid homeostasis, and they shed light on the roles of skeletal muscle in the pathogenesis of hyperlipidemia and hepatosteatosis.
Assuntos
Fígado Gorduroso/metabolismo , Hiperlipidemias/metabolismo , Metabolismo dos Lipídeos/fisiologia , Músculo Esquelético/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Colesterol/biossíntese , Regulação da Expressão Gênica/fisiologia , Hiperlipidemias/sangue , Metabolismo dos Lipídeos/genética , Camundongos , Camundongos Knockout , Fator Regulador Miogênico 5/genética , Fator Regulador Miogênico 5/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfatidato Fosfatase/genética , Fosfatidato Fosfatase/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Proteínas rab de Ligação ao GTP/genéticaRESUMO
Cell death-inducing DFF45-like effector (CIDE) family proteins including Cidea, Cideb and Cidec/Fsp27 are expressed in many different tissues and are known as lipid droplet (LD)-and ER-associated proteins. Systematic analyses using genetically modified animal models have demonstrated that CIDE proteins play important roles in regulating various aspects of lipid homeostasis, including lipid storage, lipolysis and lipid secretion. Recent research in ours and other laboratories has revealed that CIDE proteins are crucial regulators of LD fusion and growth in the adipose tissue, liver, skin and mammary glands. CIDE-mediated LD fusion and growth is different from other membrane fusions in that it requires CIDE proteins to be enriched and clustered at the LD-LD contact sites (LDCS). The enriched CIDE proteins then allow the recruitment of other proteins to the LDCS and the formation of potential fusion pores. Neutral lipids in the smaller LDs of the contacted pair are transferred to the larger LDs, owing to the internal pressure difference, thus resulting in the fusion and growth of the LDs. This review summarizes the physiological roles of CIDE proteins in controlling lipid homeostasis, insulin sensitivity and the development of metabolic diseases including obesity, diabetes and fatty liver, with a particular focus on the role of CIDE proteins in controlling LD fusion and growth. This article is part of a Special Issue entitled: Recent Advances in Lipid Droplet Biology edited by Rosalind Coleman and Matthijs Hesselink.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Gotículas Lipídicas/metabolismo , Metabolismo dos Lipídeos , Fusão de Membrana , Animais , Proteínas Reguladoras de Apoptose/genética , Diabetes Mellitus/genética , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patologia , Fígado Gorduroso/genética , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Humanos , Resistência à Insulina , Gotículas Lipídicas/patologia , Obesidade/genética , Obesidade/metabolismo , Obesidade/patologiaRESUMO
Lipid droplets (LDs) are dynamic subcellular organelles whose growth is closely linked to obesity and hepatic steatosis. Cell death-inducing DNA fragmentation factor-α-like effector (CIDE) proteins, including Cidea, Cideb, and Cidec (also called Fsp27), play important roles in lipid metabolism. Cidea and Cidec are LD-associated proteins that promote atypical LD fusion in adipocytes. Here, we find that CIDE proteins are all localized to LD-LD contact sites (LDCSs) and promote lipid transfer, LD fusion, and growth in hepatocytes. We have identified two types of hepatocytes, one with small LDs (small LD-containing hepatocytes, SLHs) and one with large LDs (large LD-containing hepatocytes, LLHs) in the liver. Cideb is localized to LDCSs and promotes lipid exchange and LD fusion in both SLHs and LLHs, whereas Cidea and Cidec are specifically localized to the LDCSs and promote lipid exchange and LD fusion in LLHs. Cideb-deficient SLHs have reduced LD sizes and lower lipid exchange activities. Fasting dramatically induces the expression of Cidea/Cidec and increases the percentage of LLHs in the liver. The majority of the hepatocytes from the liver of obese mice are Cidea/Cidec-positive LLHs. Knocking down Cidea or Cidec significantly reduced lipid storage in the livers of obese animals. Our data reveal that CIDE proteins play differential roles in promoting LD fusion and lipid storage; Cideb promotes lipid storage under normal diet conditions, whereas Cidea and Cidec are responsible for liver steatosis under fasting and obese conditions.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Fígado Gorduroso/etiologia , Hepatócitos/metabolismo , Gotículas Lipídicas/patologia , Obesidade/patologia , Proteínas/metabolismo , Animais , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Proteínas Reguladoras de Apoptose/genética , Linhagem Celular , Células Cultivadas , Privação de Alimentos , Hepatócitos/citologia , Hepatócitos/patologia , Hepatócitos/ultraestrutura , Humanos , Gotículas Lipídicas/ultraestrutura , Fusão de Membrana , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Knockout , Camundongos Obesos , Obesidade/metabolismo , Obesidade/fisiopatologia , Biogênese de Organelas , Tamanho das Organelas , Perilipina-2 , Transporte Proteico , Proteínas/antagonistas & inibidores , Proteínas/genética , Interferência de RNA , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Rab GTPases, by targeting to specific membrane compartments, play essential roles in membrane trafficking. Lipid droplets (LDs) are dynamic subcellular organelles whose growth is closely linked to obesity and hepatic steatosis. Fsp27 is shown to be required for LD fusion and growth by enriching at LD-LD contact sites. Here, we identify Rab8a as a direct interactor and regulator of Fsp27 in mediating LD fusion in adipocytes. Knockdown of Rab8a in the livers of ob/ob mice results in the accumulation of smaller LDs and lower hepatic lipid levels. Surprisingly, it is the GDP-bound form of Rab8a that exhibits fusion-promoting activity. We further discover AS160 as the GTPase activating protein (GAP) for Rab8a, which forms a ternary complex with Fsp27 and Rab8a to positively regulate LD fusion. MSS4 antagonizes Fsp27-mediated LD fusion activity through Rab8a. Our results have thus revealed a mechanistic signaling circuit controlling LD fusion and fatty liver formation.
Assuntos
Adipócitos/metabolismo , Grânulos Citoplasmáticos/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Metabolismo dos Lipídeos , Chaperonas Moleculares/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Adipócitos/citologia , Animais , Camundongos , Camundongos Obesos , Chaperonas Moleculares/genética , Células NIH 3T3 , Ligação Proteica , Proteínas/metabolismoRESUMO
Mature white adipocytes contain a characteristic unilocular lipid droplet. However, the molecular mechanisms underlying unilocular lipid droplet formation are poorly understood. We previously showed that Fsp27, an adipocyte-specific lipid droplet-associated protein, promotes lipid droplet growth by initiating lipid exchange and transfer. Here, we identify Perilipin1 (Plin1), another adipocyte-specific lipid droplet-associated protein, as an Fsp27 activator. Plin1 interacts with the CIDE-N domain of Fsp27 and markedly increases Fsp27-mediated lipid exchange, lipid transfer and lipid droplet growth. Functional cooperation between Plin1 and Fsp27 is required for efficient lipid droplet growth in adipocytes, as depletion of either protein impairs lipid droplet growth. The CIDE-N domain of Fsp27 forms homodimers and disruption of CIDE-N homodimerization abolishes Fsp27-mediated lipid exchange and transfer. Interestingly, Plin1 can restore the activity of CIDE-N homodimerization-defective mutants of Fsp27. We thus uncover a novel mechanism underlying lipid droplet growth and unilocular lipid droplet formation that involves the cooperative action of Fsp27 and Plin1 in adipocytes.
Assuntos
Adipócitos/metabolismo , Proteínas de Transporte/metabolismo , Lipídeos/química , Fosfoproteínas/metabolismo , Proteínas/metabolismo , Células 3T3-L1 , Sequência de Aminoácidos , Animais , Western Blotting , Proteínas de Transporte/química , Cristalografia por Raios X , Recuperação de Fluorescência Após Fotodegradação , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Mutação/genética , Perilipina-1 , Fosfoproteínas/química , Ligação Proteica , Multimerização Proteica , Estrutura Terciária de Proteína , Proteínas/químicaRESUMO
Lipid droplets (LDs) are dynamic cellular organelles that control many biological processes. However, molecular components determining LD growth are poorly understood. Genetic analysis has indicated that Fsp27, an LD-associated protein, is important in controlling LD size and lipid storage in adipocytes. In this paper, we demonstrate that Fsp27 is focally enriched at the LD-LD contacting site (LDCS). Photobleaching revealed the occurrence of lipid exchange between contacted LDs in wild-type adipocytes and Fsp27-overexpressing cells but not Fsp27-deficient adipocytes. Furthermore, live-cell imaging revealed a unique Fsp27-mediated LD growth process involving a directional net lipid transfer from the smaller to larger LDs at LDCSs, which is in accordance with the biophysical analysis of the internal pressure difference between the contacting LD pair. Thus, we have uncovered a novel molecular mechanism of LD growth mediated by Fsp27.
Assuntos
Adipócitos/metabolismo , Metabolismo dos Lipídeos , Organelas/metabolismo , Proteínas/metabolismo , Células 3T3 , Animais , Células HEK293 , Humanos , Camundongos , Proteínas/genéticaRESUMO
By the methods of acute toxicity test and single cell gel electrophoresis (comet assay), this paper evaluated the toxicological effects of three veterinary drugs olaquindox, arsanilic acid and oxytetracycline on earthworm (Eisenia foetida) coelomocytes in vivo. The results of acute toxicity test showed that only the highest dose of olaquidox caused the death of some earthworms, and none of the test drugs had any effects on earthworm at their environmentally relevant concentrations. The comet assay indicated that arsanilic acid had no genotoxicity to earthworm, while olaquindox and oxytetracycline induced significant DNA damage in earthworm coelomocytes (P < 0.01).