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1.
BMC Biotechnol ; 13: 68, 2013 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-24128347

RESUMO

BACKGROUND: The yeast Saccharomyces cerevisiae can be a useful model for studying cellular mechanisms related to sterol synthesis in humans due to the high similarity of the mevalonate pathway between these organisms. This metabolic pathway plays a key role in multiple cellular processes by synthesizing sterol and nonsterol isoprenoids. Statins are well-known inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the key enzyme of the cholesterol synthesis pathway. However, the effects of statins extend beyond their cholesterol-lowering action, since inhibition of HMGR decreases the synthesis of all products downstream in the mevalonate pathway. Using transgenic yeast expressing human HMGR or either yeast HMGR isoenzyme we studied the effects of simvastatin, atorvastatin, fluvastatin and rosuvastatin on the cell metabolism. RESULTS: Statins decreased sterol pools, prominently reducing sterol precursors content while only moderately lowering ergosterol level. Expression of genes encoding enzymes involved in sterol biosynthesis was induced, while genes from nonsterol isoprenoid pathways, such as coenzyme Q and dolichol biosynthesis or protein prenylation, were diversely affected by statin treatment. Statins increased the level of human HMGR protein substantially and only slightly affected the levels of Rer2 and Coq3 proteins involved in non-sterol isoprenoid biosynthesis. CONCLUSION: Statins influence the sterol pool, gene expression and protein levels of enzymes from the sterol and nonsterol isoprenoid biosynthesis branches and this effect depends on the type of statin administered. Our model system is a cheap and convenient tool for characterizing individual statins or screening for novel ones, and could also be helpful in individualized selection of the most efficient HMGR inhibitors leading to the best response and minimizing serious side effects.


Assuntos
Hidroximetilglutaril-CoA Redutases/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Ácido Mevalônico/metabolismo , Saccharomyces cerevisiae/metabolismo , Atorvastatina , Ácidos Graxos Monoinsaturados/farmacologia , Fluorbenzenos/farmacologia , Fluvastatina , Proteínas Fúngicas/metabolismo , Ácidos Heptanoicos/farmacologia , Humanos , Hidroximetilglutaril-CoA Redutases/genética , Indóis/farmacologia , Isoenzimas/metabolismo , Organismos Geneticamente Modificados , Pirimidinas/farmacologia , Pirróis/farmacologia , Rosuvastatina Cálcica , Saccharomyces cerevisiae/crescimento & desenvolvimento , Sinvastatina/farmacologia , Esteróis/biossíntese , Sulfonamidas/farmacologia , Terpenos/metabolismo
2.
Postepy Biochem ; 58(3): 344-52, 2012.
Artigo em Polonês | MEDLINE | ID: mdl-23373419

RESUMO

Mitochondria are organelles present in all eukaryotic organisms. Their primary function is production of energy in the form of ATP by oxidative phosphorylation. The final step of this process is catalyzed by an enzyme of internal mitochondrial membrane - ATP synthase. The ATP synthase consists of the seventeen subunits in yeast (in vertebrate sixteen is identified to date) organized in hydrophobic, membrane localized unit, referred to as F0 and hydrophilic domain F1 directed into mitochondria matrix. Genes encoding the ATP synthase subunits are mainly nuclear, but few of them, encoding hydrophobic subunits, are retained in mitochondrial genome in most Eukaryotes. Biogenesis of the ATP synthase is a sophisticated process, depending on the activity of proteins, which are not ATP synthase subunits, coordinating expression of the nuclear and mitochondrial genes and their assembly in active complex. This review summarizes the present knowledge about structure, biogenesis and mechanism of ATP synthase complex function.


Assuntos
ATPases Mitocondriais Próton-Translocadoras/química , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Humanos , ATPases Mitocondriais Próton-Translocadoras/genética , Fosforilação Oxidativa
3.
Protein Expr Purif ; 71(1): 28-32, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20093188

RESUMO

Plasma retinol-binding protein (RBP4) is the principal carrier of vitamin A in blood. Recent studies have suggested that RBP4 may have also a role in insulin resistance. To date the recombinant protein is usually produced by refolding inclusion bodies in Escherichia coli. Here we report the expression and characterization of recombinant human plasma RBP4 using the Pichia pastoris expression system. Simple and rapid purification allowed us to obtain 5mg/L of purified protein from the fermentation supernatant with no need to perform denaturing and refolding steps. The identity of the protein was verified by ion-trap MS and Western blotting. The functionality of recombinant RBP4, i.e., the binding to its physiologic ligand, retinol, and the interaction with transthyretin (TTR), was tested by fluorimetric and pull-down assays, respectively. The apparent dissociation constant for retinol to the recombinant protein of 2 x 10(-7)M was consistent with published data for native human protein. The recombinant protein interacted specifically with TTR. These results suggest that expression of recombinant human RBP4 in P. pastoris provides an efficient source of fully functional protein in soluble form for biochemical and biophysical studies.


Assuntos
Bioquímica/métodos , Pichia/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Plasmáticas de Ligação ao Retinol/metabolismo , Sequência de Aminoácidos , Bioensaio , Humanos , Espectrometria de Massas , Dados de Sequência Molecular , Pré-Albumina/metabolismo , Ligação Proteica , Proteínas Recombinantes/isolamento & purificação , Proteínas Plasmáticas de Ligação ao Retinol/química , Proteínas Plasmáticas de Ligação ao Retinol/isolamento & purificação , Fatores de Tempo , Vitamina A/metabolismo
4.
Eur Biophys J ; 39(4): 679-88, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19921174

RESUMO

Peripherin/RDS is a member of the tetraspanin family of integral membrane proteins and plays a major role in the morphology of photoreceptor outer segments. Peripherin/RDS has a long extracellular loop (hereafter referred to as the LEL domain), which is vital for its function. Point mutations in the LEL domain often lead to impaired photoreceptor formation and function, making peripherin/RDS an important drug target. Being a eukaryotic membrane protein, acquiring sufficient peripherin/RDS for biophysical characterisation represents a significant challenge. Here, we describe the expression and characterisation of peripherin/RDS in Drosophila melangolaster Schneider (S2) insect cells and in the methylotrophic yeast Pichia pastoris. The wild-type peripherin/RDS and the retinitis pigmentosa causing P216L mutant from S2 cells are characterised using circular dichroism (CD) spectroscopy. The structure of peripherin/RDS and of a pathogenic mutant is assessed spectroscopically for the first time. These findings are evaluated in relation to a three-dimensional model of the functionally important LEL domain obtained by protein threading.


Assuntos
Proteínas de Filamentos Intermediários/química , Proteínas de Filamentos Intermediários/genética , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Segmento Externo das Células Fotorreceptoras da Retina/metabolismo , Sequência de Aminoácidos , Animais , Células Cultivadas , Dicroísmo Circular , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Eletroforese em Gel de Poliacrilamida , Expressão Gênica , Humanos , Proteínas de Filamentos Intermediários/biossíntese , Proteínas de Filamentos Intermediários/metabolismo , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/metabolismo , Periferinas , Pichia/genética , Mutação Puntual , Estrutura Terciária de Proteína , Análise Espectral
5.
FEBS Lett ; 581(28): 5371-6, 2007 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-17967424

RESUMO

Pan1 is an actin patch-associated protein involved in endocytosis. Our studies revealed that in oleate-grown cells Pan1 is located in the nucleus as well as in patches. One of three putative nuclear localization signals (NLS) of Pan1, NLS2, directed beta-galactosidase (beta-gal) to the nucleus. However, GFP-Pan1(886-1219), containing NLS2, was found in the cytoplasm indicating that it may contain a nuclear export signal (NES). A putative Pan1 NES, overlapping with NLS3, re-addressed NLS(H2B)-NES/NLS3-beta-gal from the nucleus to the cytoplasm. Inactivation of the NES allowed NLS3 to be effective. Thus, Pan1 contains functional NLSs and a NES and appears to shuttle in certain circumstances.


Assuntos
Núcleo Celular/metabolismo , Proteínas Fúngicas/metabolismo , Sinais de Exportação Nuclear , Saccharomyces cerevisiae/metabolismo , Transporte Ativo do Núcleo Celular , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Genes Reporter/genética , Proteínas dos Microfilamentos , Dados de Sequência Molecular , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae
6.
PLoS One ; 8(9): e74187, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24073201

RESUMO

The ADP/ATP carrier protein (AAC) expressed in Artemia franciscana is refractory to bongkrekate. We generated two strains of Saccharomyces cerevisiae where AAC1 and AAC3 were inactivated and the AAC2 isoform was replaced with Artemia AAC containing a hemagglutinin tag (ArAAC-HA). In one of the strains the suppressor of ΔAAC2 lethality, SAL1, was also inactivated but a plasmid coding for yeast AAC2 was included, because the ArAACΔsal1Δ strain was lethal. In both strains ArAAC-HA was expressed and correctly localized to the mitochondria. Peptide sequencing of ArAAC expressed in Artemia and that expressed in the modified yeasts revealed identical amino acid sequences. The isolated mitochondria from both modified strains developed 85% of the membrane potential attained by mitochondria of control strains, and addition of ADP yielded bongkrekate-sensitive depolarizations implying acquired sensitivity of ArAAC-mediated adenine nucleotide exchange to this poison, independent from SAL1. However, growth of ArAAC-expressing yeasts in glycerol-containing media was arrested by bongkrekate only in the presence of SAL1. We conclude that the mitochondrial environment of yeasts relying on respiratory growth conferred sensitivity of ArAAC to bongkrekate in a SAL1-dependent manner.


Assuntos
Artemia/metabolismo , Ácido Bongcréquico/farmacologia , Farmacorresistência Fúngica , Translocases Mitocondriais de ADP e ATP/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Antibacterianos/farmacologia , Artemia/efeitos dos fármacos , Artemia/crescimento & desenvolvimento , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Translocases Mitocondriais de ADP e ATP/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética
7.
Eur J Cell Biol ; 90(10): 834-43, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21868125

RESUMO

The Rsp5 ubiquitin ligase regulates numerous cellular processes. Rsp5 is mainly localized to the cytoplasm but nuclear localization was also reported. A potential nuclear export signal was tested for activity by using a GFP(2) reporter. The 687-LIGGIAEIDI-696 sequence located in the Hect domain was identified as a nuclear export signal active in a Crm1-dependent manner, and its importance for the localization of Rsp5 was documented by using fluorescence microscopy and a lacZ-based reporter system. Analysis of the cellular location of other Rsp5 fragments fused with GFP(2) indicated two independent potential nuclear localization signals, both located in the Hect domain. We also uncovered Rsp5 fragments that are important to targeting/tethering Rsp5 to various regions in the cytoplasm. The presented data indicate that Rsp5 ligase is a shuttling protein whose distribution within the cytoplasm and partitioning between cytoplasmic and nuclear locations is determined by a balance between the actions of several targeting sequences and domains.


Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Sinais de Exportação Nuclear/genética , Sinais de Localização Nuclear/genética , Transporte Proteico , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Sequência de Aminoácidos , Núcleo Celular/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/química , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Técnicas de Inativação de Genes , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Carioferinas/genética , Carioferinas/metabolismo , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Estrutura Terciária de Proteína , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Complexos Ubiquitina-Proteína Ligase/química , Complexos Ubiquitina-Proteína Ligase/genética , Proteína Exportina 1
8.
PLoS One ; 4(12): e8499, 2009 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-20041128

RESUMO

In humans, defects in lipid metabolism are associated with a number of severe diseases such as atherosclerosis, obesity and type II diabetes. Hypercholesterolemia is a primary risk factor for coronary artery disease, the major cause of premature deaths in developed countries. Statins are inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the key enzyme of the sterol synthesis pathway. Since yeast Saccharomyces cerevisiae harbours many counterparts of mammalian enzymes involved in lipid-synthesizing pathways, conclusions drawn from research with this single cell eukaryotic organism can be readily applied to higher eukaryotes. Using a yeast strain with deletions of both HMG1 and HMG2 genes (i.e. completely devoid of HMGR activity) with introduced wild-type or mutant form of human HMGR (hHMGR) gene we investigated the effects of statins on the lipid metabolism of the cell. The relative quantification of mRNA demonstrated a different effect of simvastatin on the expression of the wild-type and mutated hHMGR gene. GC/MS analyses showed a significant decrease of sterols and enhanced conversion of squalene and sterol precursors into ergosterol. This was accompanied by the mobilization of ergosterol precursors localized in lipid particles in the form of steryl esters visualized by confocal microscopy. Changes in the level of ergosterol and its precursors in cells treated with simvastatin depend on the mutation in the hHMGR gene. HPLC/MS analyses indicated a reduced level of phospholipids not connected with the mevalonic acid pathway. We detected two significant phenomena. First, cells treated with simvastatin develop an adaptive response compensating the lower activity of HMGR. This includes enhanced conversion of sterol precursors into ergosterol, mobilization of steryl esters and increased expression of the hHMGR gene. Second, statins cause a substantial drop in the level of glycerophospholipids.


Assuntos
Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Cromatografia em Camada Fina , Cromatografia Gasosa-Espectrometria de Massas , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Humanos , Hidroximetilglutaril-CoA Redutases/genética , Hidroximetilglutaril-CoA Redutases/metabolismo , Microscopia Confocal , Proteínas Mutantes/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Sinvastatina/farmacologia , Esqualeno/metabolismo , Coloração e Rotulagem , Esteróis/metabolismo
9.
Exp Cell Res ; 310(2): 482-92, 2005 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-16171804

RESUMO

Pan1p is a yeast actin cytoskeleton-associated protein localized in actin patches. It activates the Arp2/3 complex, which is necessary for actin polymerization and endocytosis. We isolated the pan1-11 yeast mutant unable to grow on oleate as a sole carbon source and, therefore, exhibiting the Oleate- phenotype. In addition, mutant cells are temperature-sensitive and grow more slowly on glycerol or succinate-containing medium but similarly to the wild type on ethanol, pyruvate or acetate-containing media; this indicates proper functioning of the mitochondrial respiratory chain. However, growth on ethanol medium is compromised when oleic acid is present. Cells show growth arrest in the apical growth phase, and accumulation of cells with abnormally elongated buds is observed. The growth defects of pan1-11 are suppressed by overexpression of the END3 gene encoding a protein that binds Pan1p. The morphology of peroxisomes and induction of peroxisomal enzymes are normal in pan1-11, indicating that the defect in growth on oleate medium does not result from impairment in peroxisome function. The pan1-11 allele has a deletion of a fragment encoding amino acids 1109-1126 that are part of (QPTQPV)7 repeats. Surprisingly, the independently isolated pan1-9 mutant, which expresses a truncated form of Pan1p comprising aa 1-859, is able to grow on all media tested. Our results indicate that Pan1p, and possibly other components of the actin cytoskeleton, are necessary to properly regulate growth of dividing cells in response to the presence of some alternative carbon sources in the medium.


Assuntos
Proteínas Fúngicas/genética , Ácido Oleico/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/genética , Actinas/química , Actinas/metabolismo , Alelos , Parede Celular/genética , Parede Celular/metabolismo , Parede Celular/ultraestrutura , Meios de Cultura/metabolismo , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/genética , Citoesqueleto/metabolismo , Endocitose/genética , Proteínas Fúngicas/análise , Proteínas Fúngicas/metabolismo , Proteínas dos Microfilamentos , Mutação , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/análise , Proteínas de Saccharomyces cerevisiae/metabolismo
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