RESUMO
Our experiments aim to determine if decreasing the amount of phosphatidylcholine (PC) relative to phosphatidylethanolamine (PE) at the lipid droplet surface changes the localization of specific lipid droplet proteins. We manipulate lipid droplet phospholipids in both a cultured mouse hepatocyte (AML12) cell line and on synthetic lipid droplets. Decreasing the PC:PE ratio increases perilipin 2, decreases DGAT2, and does not change rab18 or lanosterol synthase levels on lipid droplets. These differences may be explained by the distinct structural motifs that mediate the protein-lipid droplet interactions.
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BACKGROUND: The earliest manifestation of alcohol-related liver disease (ALD) is steatosis, characterized by the accumulation of lipid droplets (LDs) in hepatocytes. Findings from our laboratory have indicated that many pathological changes, including steatosis, correlate with the alcohol-induced hepatocellular increases in S-adenosylhomocysteine (SAH). Based on these considerations, we hypothesized that an experimental increase in intracellular SAH alone will result in similar steatotic changes to those seen after alcohol exposure. METHODS: Freshly isolated rat hepatocytes grown on collagen-coated plates were exposed to serum-free medium containing 50 µmol/L oleic acid and varying concentrations of 3-deazaadenosine (DZA) to experimentally elevate intracellular SAH levels. RESULTS: Overnight exposure to DZA treatment dose-dependently increased hepatocellular triglyceride accumulation, which was also evident by morphological visualization of larger-sized LDs. The rise in triglycerides and LDs accompanied increases in mRNA and protein levels of several LD-associated proteins known to regulate LD number and size. Furthermore, DZA treatment caused a decline in the levels of lipases that prevent fat accumulation as well as increased the expression of factors involved in lipogenesis and fatty acid mobilization. Collectively, our results indicate that the elevation of intracellular SAH is sufficient to promote fat accumulation in hepatocytes, which is similar to that seen after alcohol exposure.
Assuntos
Antibióticos Antineoplásicos/uso terapêutico , Etanol/efeitos adversos , Metabolismo dos Lipídeos/efeitos dos fármacos , Hepatopatias/sangue , Hepatopatias/etiologia , S-Adenosil-Homocisteína/efeitos adversos , Tubercidina/uso terapêutico , Animais , Antibióticos Antineoplásicos/farmacologia , Humanos , Hepatopatias/patologia , Masculino , Ratos , Ratos Wistar , Tubercidina/farmacologiaRESUMO
Alcoholic fatty liver disease (AFLD) is characterized by an abnormal accumulation of lipid droplets (LDs) in the liver. Here, we explore the composition of hepatic LDs in a rat model of AFLD. Five to seven weeks of alcohol consumption led to significant increases in hepatic triglyceride mass, along with increases in LD number and size. Additionally, hepatic LDs from rats with early alcoholic liver injury show a decreased ratio of surface phosphatidylcholine (PC) to phosphatidylethanolamine (PE). This occurred in parallel with an increase in the LD association of perilipin 2, a prominent LD protein. To determine if changes to the LD phospholipid composition contributed to differences in protein association with LDs, we constructed liposomes that modeled the LD PC:PE ratios in AFLD and control rats. Reducing the ratio of PC to PE increased the binding of perilipin 2 to liposomes in an in vitro experiment. Moreover, we decreased the ratio of LD PC:PE in NIH 3T3 and AML12 cells by culturing these cells in choline-deficient media. We again detected increased association of specific LD proteins, including perilipin 2. Taken together, our experiments suggest an important link between LD phospholipids, protein composition, and lipid accumulation.
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Recognizing that undergraduate students can benefit from analysis of 3D protein structure and function, we have developed a multiweek, inquiry-based molecular visualization project for Biochemistry I students. This project uses a virtual model of cyclooxygenase-1 (COX-1) to guide students through multiple levels of protein structure analysis. The first assignment explores primary structure by generating and examining a protein sequence alignment. Subsequent assignments introduce 3D visualization software to explore secondary, tertiary, and quaternary structure. Students design an inhibitor, based on scrutiny of the enzyme active site, and evaluate the fit of the molecule using computed binding energies. In the last assignment, students introduce a point mutation to model the active site of the related COX-2 enzyme and analyze the impact of the mutation on inhibitor binding. With this project we aim to increase knowledge about, and confidence in using, online databases and computational tools. Here, we share results of our mixed methods pre- and postsurvey demonstrating student gains in knowledge about, and confidence using, online databases and computational tools. © 2017 by The International Union of Biochemistry and Molecular Biology, 45(4):318-328, 2017.
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Bioquímica/educação , Educação de Graduação em Medicina/métodos , Avaliação Educacional/estatística & dados numéricos , Biologia Molecular/educação , Software , Sequência de Aminoácidos , Domínio Catalítico , Ciclo-Oxigenase 1/química , Ciclo-Oxigenase 2/química , Inibidores de Ciclo-Oxigenase/química , Desenho de Fármacos , Humanos , Modelos Moleculares , Mutação Puntual , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Alinhamento de Sequência , Relação Estrutura-Atividade , Estudantes , TermodinâmicaRESUMO
A thorough understanding of the molecular biosciences requires the ability to visualize and manipulate molecules in order to interpret results or to generate hypotheses. While many instructors in biochemistry and molecular biology use visual representations, few indicate that they explicitly teach visual literacy. One reason is the need for a list of core content and competencies to guide a more deliberate instruction in visual literacy. We offer here the second stage in the development of one such resource for biomolecular three-dimensional visual literacy. We present this work with the goal of building a community for online resource development and use. In the first stage, overarching themes were identified and submitted to the biosciences community for comment: atomic geometry; alternate renderings; construction/annotation; het group recognition; molecular dynamics; molecular interactions; monomer recognition; symmetry/asymmetry recognition; structure-function relationships; structural model skepticism; and topology and connectivity. Herein, the overarching themes have been expanded to include a 12th theme (macromolecular assemblies), 27 learning goals, and more than 200 corresponding objectives, many of which cut across multiple overarching themes. The learning goals and objectives offered here provide educators with a framework on which to map the use of molecular visualization in their classrooms. In addition, the framework may also be used by biochemistry and molecular biology educators to identify gaps in coverage and drive the creation of new activities to improve visual literacy. This work represents the first attempt, to our knowledge, to catalog a comprehensive list of explicit learning goals and objectives in visual literacy. © 2016 by The International Union of Biochemistry and Molecular Biology, 45(1):69-75, 2017.
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Bioquímica/educação , Gráficos por Computador , Processamento de Imagem Assistida por Computador/métodos , Aprendizagem , Biologia Molecular/educação , Imagem Molecular/métodos , Proteínas/química , Objetivos , Humanos , Modelos Educacionais , Modelos Moleculares , EstudantesRESUMO
Excess lipid is stored in intracellular organelles known as lipid droplets. This unit discusses techniques for the visualization of lipid droplets and associated proteins in cultured mammalian cells. Protocols for the detection of lipid droplets in fixed or live cells with BODIPY 493/503 are included. The best method for combining visualization of intracellular lipid droplets with indirect immunofluorescent detection of lipid droplet-associated proteins is described. Techniques for sample fixation and permeabilization must be chosen carefully to avoid alterations to lipid droplet morphology. Immunofluorescent detection of perilipin 2, a broadly expressed, lipid droplet-associated protein, widely used as a marker for lipid droplet accumulation, is presented as an example. Finally, a simple protocol for enhancing lipid droplet accumulation through supplementation with excess fatty acid is included. © 2016 by John Wiley & Sons, Inc.
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Imunofluorescência/métodos , Gotículas Lipídicas/química , Proteínas de Membrana/análise , Compostos de Boro , Linhagem Celular Tumoral , Humanos , Perilipina-2/metabolismoRESUMO
All eukaryotic organisms store excess lipid in intracellular lipid droplets. These dynamic structures are associated with and regulated by numerous proteins. Perilipin 2, an abundant protein on most lipid droplets, promotes neutral lipid accumulation in lipid droplets. However, the mechanism by which perilipin 2 binds to and remains anchored on the lipid droplet surface is unknown. Here we identify features of the lipid droplet surface that influence perilipin 2 localization. We show that perilipin 2 binding to the lipid droplet surface requires both hydrophobic and electrostatic interactions. Reagents that disrupt these interactions also decrease binding. Moreover, perilipin 2 binding does not depend on other lipid droplet-associated proteins but is influenced by the lipid composition of the surface. Perilipin 2 binds to synthetic vesicles composed of dioleoylphosphatidylcholine, a phospholipid with unsaturated acyl chains. Decreasing the temperature of the binding reaction, or introducing phospholipids with saturated acyl chains, decreases binding. We therefore demonstrate a role for surface lipids and acyl chain packing in perilipin 2 binding to lipid droplets. The ability of the lipid droplet phospholipid composition to impact protein binding may link changes in nutrient availability to lipid droplet homeostasis.
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Gotículas Lipídicas/química , Lipossomos/química , Proteínas de Membrana/química , Fosfatidilcolinas/química , Sítios de Ligação , Expressão Gênica , Células HEK293 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Gotículas Lipídicas/metabolismo , Lipossomos/metabolismo , Proteínas de Membrana/metabolismo , Perilipina-2 , Fosfatidilcolinas/metabolismo , Ligação Proteica , Proteólise , Eletricidade EstáticaRESUMO
Perilipin 2 (PLIN2) is the most abundant lipid droplet (LD)-associated protein in nonadipose tissue, and its expression correlates with intracellular lipid accumulation. Here we identified a missense polymorphism, Ser251Pro, that has major effect on protein structure and function, along with an influence on human plasma triglyceride concentration. The evolutionarily conserved Ser251Pro polymorphism was identified with the ClustalW program. Structure modeling using 3D-JigSaw and the Chimera package revealed that the Pro251 allele disrupts a predicted α-helix in PLIN2. Analyses of macrophages from individuals carrying Ser251Pro variants and human embryonic kidney 293 (HEK293) cells stably transfected with either of the alleles demonstrated that the Pro251 variant causes increased lipid accumulation and decreased lipolysis. Analysis of LD size distribution in stably transfected cells showed that the minor Pro251 allele resulted in an increased number of small LDs per cell and increased perilipin 3 protein expression levels as compared with cells carrying the major Ser251 allele. Genotyping of 2113 individuals indicated that the Pro251 variant is associated with decreased plasma triglyceride and very low-density lipoprotein concentrations. Altogether, these data provide the first evidence of a polymorphism in PLIN2 that affects PLIN2 function and may influence the development of metabolic and cardiovascular diseases.
Assuntos
Lipólise/genética , Proteínas de Membrana/genética , Mutação de Sentido Incorreto , Polimorfismo Genético , Triglicerídeos/sangue , Adulto , Alelos , Sequência de Aminoácidos , Células Cultivadas , Grânulos Citoplasmáticos/metabolismo , Grânulos Citoplasmáticos/ultraestrutura , Feminino , Genótipo , Células HEK293 , Humanos , Lipídeos/análise , Lipoproteínas VLDL/sangue , Masculino , Proteínas de Membrana/química , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Pessoa de Meia-Idade , Modelos Moleculares , Dados de Sequência Molecular , Perilipina-2 , Estrutura Secundária de Proteína/genética , Homologia de Sequência de Aminoácidos , Triglicerídeos/metabolismoRESUMO
BACKGROUND: In the renal collecting duct, vasopressin regulates water permeability by a process that involves stimulation of adenylyl cyclase activity, cAMP production and subsequent translocation of water channel aquaporin-2 (AQP2) into the apical plasma membrane. We have previously shown that in cos 1 cells in vitro, both adenylyl cyclase activity and cAMP production can be regulated by VACM-1, a cul 5 gene that forms complexes involved in protein ubiquitination and subsequent degradation. METHODS: To extend these observations further, the effects of changes in hydration state on the expression of VACM-1 at the mRNA and the protein level were examined in rats deprived of water (WD) for 24 hrs. RESULTS: In the kidney of WD rats Western blot analyses of kidney tissue showed that the decrease in VACM-1 protein concentration was correlated with the increase in the AQP2 protein level. The immunostaining data suggested that VACM-1/cul5 may be decreased in renal collecting duct but increases in the vasculature of the inner medullary region in response to WD. To determine the possible consequences of the WD dependent decrease in VACM-1/cul5, we next examined the effects of VACM-1 expression on AQP2 protein in vitro. Immunocytochemistry and Western blot analyses data indicate that VACM-1/cul5 expression in MDCK line stably expressing AQP2 gene and in cos 1 cells co-transfected with the AQP2 and VACM-1/cul5 cDNAs decreased AQP2 protein concentration when compared to the vector transfected control groups. CONCLUSION: In summary, our data demonstrate that VACM-1 is involved in the regulation of AQP2 protein concentration and may play a role in regulating water balance.
Assuntos
Aquaporina 2/análise , Proteínas Culina/metabolismo , Receptores de Vasopressinas/metabolismo , Animais , Aquaporina 2/genética , Aquaporina 2/metabolismo , Células COS , Células Cultivadas , Chlorocebus aethiops , Proteínas Culina/genética , Cães , Rim/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Receptores de Vasopressinas/genéticaRESUMO
Lipotoxicity is a metabolic stress response implicated in the pathogenesis of diabetes complications and has been shown to involve lipid-induced oxidative stress. To elucidate the molecular mechanisms of lipotoxicity, we used retroviral promoter trap mutagenesis to isolate a cell line that is resistant to lipotoxic and oxidative stress. We show that loss of three box C/D small nucleolar RNAs (snoRNAs) encoded in the ribosomal protein L13a (rpL13a) locus is sufficient to confer resistance to lipotoxic and oxidative stress in vitro and prevents the propagation of oxidative stress in vivo. Our results provide evidence for a previously unappreciated, non-canonical role for box C/D snoRNAs as regulators of metabolic stress response pathways in mammalian cells.
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RNA Nucleolar Pequeno/metabolismo , Estresse Fisiológico , Animais , Apoptose , Sequência de Bases , Células CHO , Cricetinae , Cricetulus , Técnicas de Silenciamento de Genes , Camundongos , Dados de Sequência Molecular , Estresse Oxidativo , Palmitatos/toxicidade , Proteínas Ribossômicas/antagonistas & inibidores , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismoRESUMO
In obesity and diabetes, an imbalance in fatty acid uptake and fatty acid utilization leads to excess accumulation of lipid in non-adipose tissues. This lipid overload is associated with cellular dysfunction and cell death, which contribute to organ failure, a phenomenon termed lipotoxicity. To elucidate the molecular mechanism of lipid-mediated cell death, we generated and characterized a mutant Chinese hamster ovary cell line that is resistant to palmitate-induced cell death. In this mutant, random insertion of a retroviral promoter trap has disrupted the gene for the non-coding RNA, growth arrested DNA-damage inducible gene 7 (gadd7). Here we report that gadd7 is induced by lipotoxic stress in a reactive oxygen species (ROS)-dependent fashion and is necessary for both lipid- and general oxidative stress-mediated cell death. Depletion of gadd7 by mutagenesis or short hairpin RNA knockdown significantly reduces lipid and non-lipid induced ROS. Furthermore, depletion of gadd7 delays and diminishes ROS-induced endoplasmic reticulum stress. Together these data are the first to implicate a non-coding RNA in a feed-forward loop with oxidative stress and its induction of the endoplasmic reticulum stress response.
Assuntos
Retículo Endoplasmático/metabolismo , Ácidos Graxos/metabolismo , Estresse Oxidativo , RNA não Traduzido/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Diabetes Mellitus/genética , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patologia , Retículo Endoplasmático/genética , Retículo Endoplasmático/patologia , Ácidos Graxos/genética , Mutagênese , Mutação , Obesidade/genética , Obesidade/metabolismo , Obesidade/patologia , RNA não Traduzido/genéticaRESUMO
Although neutral lipid storage droplets are ubiquitous in eukaryotic cells, very little is known about how their synthesis and turnover are controlled. Adipocyte differentiation-related protein (ADRP; also known as adipophilin) is found on the surface of lipid droplets in most mammalian cell types. To learn how ADRP affects lipid storage, we stably expressed the protein in human embryonic kidney 293 (HEK 293) cells, which express little endogenous ADRP. As expected, ADRP was targeted to the surface of lipid droplets and caused an increase in triacylglycerol (TAG) mass under both basal and oleate-supplemented conditions. At least part of the increased mass resulted from a 50% decrease in the rate of TAG hydrolysis in ADRP-expressing cells. Furthermore, ADRP expression increased the fraction of total cellular TAG that was stored in lipid droplets. ADRP expression induced a striking decrease in the association of adipose triglyceride lipase (ATGL) and mannose-6-phosphate receptor tail-interacting protein of 47 kDa with lipid droplets and also decreased the lipid droplet association of several other unknown proteins. Transient expression of ADRP in two other cell lines also reduced the lipid droplet association of catalytically inactive ATGL. We conclude that the reduced lipid droplet association of ATGL and/or other lipases may explain the decrease in TAG turnover observed in ADRP-expressing HEK 293 cells.
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Lipase/metabolismo , Metabolismo dos Lipídeos , Proteínas de Membrana/metabolismo , Triglicerídeos/metabolismo , Animais , Cricetinae , Humanos , Hidrólise , Camundongos , Células NIH 3T3 , Perilipina-2 , TransfecçãoRESUMO
Most eukaryotic cells can store excess lipid in cytosolic lipid droplets. This unit discusses techniques for the visualization of lipid droplets and associated proteins in cultured mammalian cells. Protocols for the detection of lipid droplets with nile red and BODIPY 493/503 are included. The differences in the spectral properties of these two lipophilic dyes and advantages of each are discussed. The best method for combining visualization of intracellular lipid droplets with indirect immunofluorescent detection of lipid droplet-associated proteins is described. Techniques for sample fixation and permeabilization must be chosen carefully to avoid alterations to lipid droplet morphology. Immunofluorescent detection of adipophilin, a broadly expressed, lipid droplet-associated protein, widely used as a marker for lipid droplet accumulation, is presented as an example. Finally, a simple protocol for enhancing lipid droplet accumulation through supplementation with excess fatty acid is included.
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Citosol/química , Células Eucarióticas/química , Técnica Indireta de Fluorescência para Anticorpo/métodos , Lipídeos/análise , Microscopia de Fluorescência/métodos , Animais , Biomarcadores , Compostos de Boro/análise , Compostos de Boro/metabolismo , Células Cultivadas/química , Células Cultivadas/ultraestrutura , Meios de Cultura/farmacologia , Células Eucarióticas/ultraestrutura , Ácidos Graxos/metabolismo , Ácidos Graxos/farmacologia , Corantes Fluorescentes/análise , Corantes Fluorescentes/metabolismo , Indicadores e Reagentes , Mamíferos/anatomia & histologia , Proteínas de Membrana , Oxazinas/análise , Oxazinas/metabolismo , Peptídeos/análise , Perilipina-2RESUMO
The deleterious consequences of fatty acid (FA) and neutral lipid accumulation in nonadipose tissues, such as the heart, contribute to the pathogenesis of type 2 diabetes. To elucidate mechanisms of FA-induced cell death, or lipotoxicity, we generated Chinese hamster ovary (CHO) cell mutants resistant to palmitate-induced death and isolated a clone with disruption of eukaryotic elongation factor (eEF) 1A-1. eEF1A-1 involvement in lipotoxicity was confirmed in H9c2 cardiomyoblasts, in which small interfering RNA-mediated knockdown also conferred palmitate resistance. In wild-type CHO and H9c2 cells, palmitate increased reactive oxygen species and induced endoplasmic reticulum (ER) stress, changes accompanied by increased eEF1A-1 expression. Disruption of eEF1A-1 expression rendered these cells resistant to hydrogen peroxide- and ER stress-induced death, indicating that eEF1A-1 plays a critical role in the cell death response to these stressors downstream of lipid overload. Disruption of eEF1A-1 also resulted in actin cytoskeleton defects under basal conditions and in response to palmitate, suggesting that eEF1A-1 mediates lipotoxic cell death, secondary to oxidative and ER stress, by regulating cytoskeletal changes critical for this process. Furthermore, our observations of oxidative stress, ER stress, and induction of eEF1A-1 expression in a mouse model of lipotoxic cardiomyopathy implicate this cellular response in the pathophysiology of metabolic disease.
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Miócitos Cardíacos/metabolismo , Palmitatos/toxicidade , Fator 1 de Elongação de Peptídeos/fisiologia , Animais , Biomarcadores/metabolismo , Cardiomiopatias/induzido quimicamente , Cardiomiopatias/metabolismo , Morte Celular , Linhagem Celular , Cricetinae , Cricetulus , Modelos Animais de Doenças , Retículo Endoplasmático/fisiologia , Camundongos , Modelos Biológicos , Mutagênese Insercional , Miócitos Cardíacos/efeitos dos fármacos , Estresse Oxidativo , Fator 1 de Elongação de Peptídeos/metabolismo , Interferência de RNA , Ratos , Espécies Reativas de Oxigênio/metabolismoRESUMO
Excess lipid accumulation in non-adipose tissues is associated with insulin resistance, pancreatic beta-cell apoptosis and heart failure. Here, we demonstrate in cultured cells that the relative toxicity of two common dietary long chain fatty acids is related to channeling of these lipids to distinct cellular metabolic fates. Oleic acid supplementation leads to triglyceride accumulation and is well tolerated, whereas excess palmitic acid is poorly incorporated into triglyceride and causes apoptosis. Unsaturated fatty acids rescue palmitate-induced apoptosis by channeling palmitate into triglyceride pools and away from pathways leading to apoptosis. Moreover, in the setting of impaired triglyceride synthesis, oleate induces lipotoxicity. Our findings support a model of cellular lipid metabolism in which unsaturated fatty acids serve a protective function against lipotoxicity though promotion of triglyceride accumulation.
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Ácidos Graxos/toxicidade , Triglicerídeos/metabolismo , Animais , Apoptose/efeitos dos fármacos , Células CHO , Linhagem Celular , Cricetinae , Resistência a Medicamentos , Ácidos Graxos Dessaturases/metabolismo , Ácidos Graxos/metabolismo , Metabolismo dos Lipídeos , Camundongos , Modelos Biológicos , Ácido Oleico/metabolismo , Ácido Oleico/farmacologia , Ácido Palmítico/metabolismo , Ácido Palmítico/toxicidadeRESUMO
The 63-kDa murine fatty acid transport protein 1 (FATP1) was cloned on the basis of its ability to augment fatty acid import when overexpressed in mammalian cells. The membrane topology of this integral plasma membrane protein does not resemble that of polytopic membrane transporters for other substrates. Western blot analysis of 3T3-L1 adipocytes that natively express FATP1 demonstrate a prominent 130-kDa species as well as the expected 63-kDa FATP1, suggesting that this protein may participate in a cell surface transport protein complex. To test whether FATP1 is capable of oligomerization, we expressed functional FATP1 molecules with different amino- or carboxyl-terminal epitope tags in fibroblasts. These epitope-tagged proteins also form apparent higher molecular weight species. We show that, when expressed in the same cells, differentially tagged FATP1 proteins co-immunoprecipitate. The region between amino acid residues 191 and 475 is sufficient for association of differentially tagged truncated FATP1 constructs. When wild type FATP1 and the non-functional s250a FATP1 mutant are co-expressed in COS7 cells, mutant FATP1 has dominant inhibitory function in fatty acid uptake assays. Taken together, these results are consistent with a model in which FATP1 homodimeric complexes play an important role in cellular fatty acid import.
Assuntos
Proteínas de Transporte/química , Proteínas de Membrana Transportadoras , Células 3T3 , Animais , Proteínas de Transporte/fisiologia , Dimerização , Proteínas de Transporte de Ácido Graxo , Ácidos Graxos/metabolismo , Camundongos , Peso Molecular , Testes de PrecipitinaRESUMO
Polypeptide binding by the chaperone Hsp70 is regulated by its ATPase activity, which is itself regulated by co-chaperones including the Bag domain nucleotide exchange factors. Here, we tested the functional contribution of residues in the Bag domain of Bag-1M that contact Hsp70. Two point mutations, E212A and E219A, partially reduced co-chaperone activity, whereas the point mutation R237A completely abolished activity in vitro. Based on the strict positional conservation of the Arg-237 residue, several Bag domain proteins were predicted from various eukaryotic genomes. One candidate, Snl1p from Saccharomyces cerevisiae, was confirmed as a Bag domain co-chaperone. Snl1p bound specifically to the Ssa and Ssb forms of yeast cytosolic Hsp70, as revealed by two-hybrid screening and co-precipitations from yeast lysate. In vitro, Snl1p also recognized mammalian Hsp70 and regulated the Hsp70 ATPase activity identically to Bag-1M. Point mutations in Snl1p that disrupted the conserved residues Glu-112 and Arg-141, equivalent to Glu-212 and Arg-237 in Bag-1M, abolished the interaction with Hsp70 proteins. In live yeast, mutated Snl1p could not substitute for wild-type Snl1p in suppressing the lethal defect caused by truncation of the Nup116p nuclear pore component. Thus, Snl1p is the first Bag domain protein identified in S. cerevisiae, and its interaction with Hsp70 is essential for biological activity.
Assuntos
Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Membrana/metabolismo , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Arginina/química , Calorimetria , Núcleo Celular/metabolismo , Citosol/metabolismo , Análise Mutacional de DNA , Ácido Glutâmico/química , Proteínas de Choque Térmico HSC70 , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Modelos Químicos , Modelos Moleculares , Chaperonas Moleculares , Dados de Sequência Molecular , Mutação , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Plasmídeos/metabolismo , Mutação Puntual , Testes de Precipitina , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas de Saccharomyces cerevisiae/metabolismo , Relação Estrutura-Atividade , Técnicas do Sistema de Duplo-HíbridoRESUMO
Accumulation of long-chain fatty acids in the heart has been proposed to play a role in the development of heart failure and diabetic cardiomyopathy. Several animal models with increased cardiomyocyte lipid accumulation suggest a link between the accumulation of lipid, cardiomyocyte cell death and the development of cardiomyopathy. In this review, we discuss the mechanism through which fatty acid accumulation may contribute to the development or progression of heart failure by initiation of apoptotic cell death. Long-chain saturated fatty acids induce apoptosis through a mechanism involving the generation of reactive intermediates. Reactive intermediate production occurs in concert with de novo ceramide synthesis, but ceramide production is not required for cell death. Cardiomyocyte dysfunction and death from reactive intermediates generated by long-chain saturated fatty acids may contribute to the pathogenesis of human heart disease.