RESUMO
Lipoprotein lipase (LPL) is the key enzyme that hydrolyzes triglycerides from triglyceride-rich lipoproteins. Angiopoietin-like proteins (ANGPTL) 3, 4, and 8 are well-characterized protein inhibitors of LPL. ANGPTL8 forms a complex with ANGPTL3, and the complex is a potent endogenous inhibitor of LPL. However, the nature of the structural interaction between ANGPTL3/8 and LPL is unknown. To probe the conformational changes in LPL induced by ANGPTL3/8, we found that HDX-MS detected significantly altered deuteration in the lid region, ApoC2 binding site, and furin cleavage region of LPL in the presence of ANGPTL3/8. Supporting this HDX structural evidence, we found that ANGPTL3/8 inhibits LPL enzymatic activities and increases LPL cleavage. ANGPTL3/8-induced effects on LPL activity and LPL cleavage are much stronger than those of ANGPTL3 or ANGPTL8 alone. ANGPTL3/8-mediated LPL cleavage is blocked by both an ANGPTL3 antibody and a furin inhibitor. Knock-down of furin expression by siRNA significantly reduced ANGPT3/8-induced cleavage of LPL. Our data suggest ANGPTL3/8 promotes furin-mediated LPL cleavage.
Assuntos
Proteínas Semelhantes a Angiopoietina/química , Lipase Lipoproteica/antagonistas & inibidores , Lipase Lipoproteica/química , Proteólise/efeitos dos fármacos , Sítios de Ligação , Deutério/química , Furina/química , Furina/genética , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Hidrólise , Marcação por Isótopo , Espectrometria de Massas , Modelos Moleculares , Ligação Proteica , Conformação Proteica , RNA Interferente Pequeno/metabolismoRESUMO
Second-harmonic generation (SHG) has recently emerged as a biophysical tool for conformational sensing of a target biomolecule upon binding to ligands such as small molecules, fragments, proteins, peptides, and oligonucleotides. To date, SHG has been used to measure conformational changes of targets such as soluble proteins, protein complexes, intrinsically disordered proteins, peripheral and integral membrane proteins, peptides, and oligonucleotides upon binding of ligands over a wide range of affinities. In this chapter, we will provide a technology overview, detailed protocols for optimizing assays and screening, practical considerations, and an example case study to guide the reader in developing robust and informative measurements using the Biodesy Delta SHG platform.
Assuntos
Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala/métodos , Oligonucleotídeos/metabolismo , Peptídeos/metabolismo , Proteínas/metabolismo , Animais , Descoberta de Drogas/instrumentação , Desenho de Equipamento , Ensaios de Triagem em Larga Escala/instrumentação , Humanos , Ligantes , Conformação de Ácido Nucleico/efeitos dos fármacos , Oligonucleotídeos/química , Peptídeos/química , Conformação Proteica/efeitos dos fármacos , Proteínas/químicaRESUMO
Activation of free fatty acid receptor 1 (GPR40) by synthetic partial and full agonists occur via distinct allosteric sites. A crystal structure of GPR40-TAK-875 complex revealed the allosteric site for the partial agonist. Here we report the 2.76-Å crystal structure of human GPR40 in complex with a synthetic full agonist, compound 1, bound to the second allosteric site. Unlike TAK-875, which acts as a Gαq-coupled partial agonist, compound 1 is a dual Gαq and Gαs-coupled full agonist. compound 1 binds in the lipid-rich region of the receptor near intracellular loop 2 (ICL2), in which the stabilization of ICL2 by the ligand is likely the primary mechanism for the enhanced G protein activities. The endogenous free fatty acid (FFA), γ-linolenic acid, can be computationally modeled in this site. Both γ-linolenic acid and compound 1 exhibit positive cooperativity with TAK-875, suggesting that this site could also serve as a FFA binding site.
Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Hipoglicemiantes/farmacologia , Incretinas/metabolismo , Secreção de Insulina , Receptores Acoplados a Proteínas G/agonistas , Sítio Alostérico/genética , Animais , Benzofuranos/farmacologia , Benzofuranos/uso terapêutico , Cristalografia por Raios X , Diabetes Mellitus Tipo 2/metabolismo , Sinergismo Farmacológico , Células HEK293 , Humanos , Hipoglicemiantes/uso terapêutico , Insulina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Knockout , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sulfonas/farmacologia , Sulfonas/uso terapêutico , Ácido gama-Linolênico/metabolismoRESUMO
SMYD2 is a lysine methyltransferase that catalyzes the monomethylation of several protein substrates including p53. SMYD2 is overexpressed in a significant percentage of esophageal squamous primary carcinomas, and that overexpression correlates with poor patient survival. However, the mechanism(s) by which SMYD2 promotes oncogenesis is not understood. A small molecule probe for SMYD2 would allow for the pharmacological dissection of this biology. In this report, we disclose LLY-507, a cell-active, potent small molecule inhibitor of SMYD2. LLY-507 is >100-fold selective for SMYD2 over a broad range of methyltransferase and non-methyltransferase targets. A 1.63-Å resolution crystal structure of SMYD2 in complex with LLY-507 shows the inhibitor binding in the substrate peptide binding pocket. LLY-507 is active in cells as measured by reduction of SMYD2-induced monomethylation of p53 Lys(370) at submicromolar concentrations. We used LLY-507 to further test other potential roles of SMYD2. Mass spectrometry-based proteomics showed that cellular global histone methylation levels were not significantly affected by SMYD2 inhibition with LLY-507, and subcellular fractionation studies indicate that SMYD2 is primarily cytoplasmic, suggesting that SMYD2 targets a very small subset of histones at specific chromatin loci and/or non-histone substrates. Breast and liver cancers were identified through in silico data mining as tumor types that display amplification and/or overexpression of SMYD2. LLY-507 inhibited the proliferation of several esophageal, liver, and breast cancer cell lines in a dose-dependent manner. These findings suggest that LLY-507 serves as a valuable chemical probe to aid in the dissection of SMYD2 function in cancer and other biological processes.
Assuntos
Antineoplásicos/química , Benzamidas/química , Inibidores Enzimáticos/química , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Neoplasias/enzimologia , Pirrolidinas/química , Linhagem Celular Tumoral , Proliferação de Células , Cromatina/química , Biologia Computacional , Cristalização , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Epigênese Genética , Histonas/química , Humanos , Espectrometria de Massas , Neoplasias/tratamento farmacológico , Peptídeos/química , Desnaturação Proteica , Proteômica , Proteína Supressora de Tumor p53/metabolismoRESUMO
The enhancer-of-zeste homolog 2 (EZH2) gene product is an 87 kDa polycomb group (PcG) protein containing a C-terminal methyltransferase SET domain. EZH2, along with binding partners, i.e., EED and SUZ12, upon which it is dependent for activity forms the core of the polycomb repressive complex 2 (PRC2). PRC2 regulates gene silencing by catalyzing the methylation of histone H3 at lysine 27. Both overexpression and mutation of EZH2 are associated with the incidence and aggressiveness of various cancers. The novel crystal structure of the SET domain was determined in order to understand disease-associated EZH2 mutations and derive an explanation for its inactivity independent of complex formation. The 2.00 Å crystal structure reveals that, in its uncomplexed form, the EZH2 C-terminus folds back into the active site blocking engagement with substrate. Furthermore, the S-adenosyl-L-methionine (SAM) binding pocket observed in the crystal structure of homologous SET domains is notably absent. This suggests that a conformational change in the EZH2 SET domain, dependent upon complex formation, must take place for cofactor and substrate binding activities to be recapitulated. In addition, the data provide a structural context for clinically significant mutations found in the EZH2 SET domain.
Assuntos
Domínio Catalítico/genética , Doença/genética , Mutação , Complexo Repressor Polycomb 2/química , Complexo Repressor Polycomb 2/metabolismo , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Proteína Potenciadora do Homólogo 2 de Zeste , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Complexo Repressor Polycomb 2/antagonistas & inibidores , Complexo Repressor Polycomb 2/genética , Células Sf9 , SpodopteraRESUMO
The sirtuin SIRT1 is a NAD(+)-dependent histone deacetylase, a Sir2 family member, and one of seven human sirtuins. Sirtuins are conserved from archaea to mammals and regulate transcription, genome stability, longevity, and metabolism. SIRT1 regulates transcription via deacetylation of transcription factors such as PPARγ, NFκB, and the tumor suppressor protein p53. EX527 (27) is a nanomolar SIRT1 inhibitor and a micromolar SIRT2 inhibitor. To elucidate the mechanism of SIRT inhibition by 27, we determined the 2.5 Å crystal structure of the SIRT1 catalytic domain (residues 241-516) bound to NAD(+) and the 27 analogue compound 35. 35 binds deep in the catalytic cleft, displacing the NAD(+) nicotinamide and forcing the cofactor into an extended conformation. The extended NAD(+) conformation sterically prevents substrate binding. The SIRT1/NAD(+)/35 crystal structure defines a novel mechanism of histone deacetylase inhibition and provides a basis for understanding, and rationally improving, inhibition of this therapeutically important target by drug-like molecules.
Assuntos
Carbazóis/farmacologia , Inibidores de Histona Desacetilases/farmacologia , NAD/metabolismo , Sirtuína 1/metabolismo , Carbazóis/química , Domínio Catalítico , Cristalografia por Raios X , Inibidores de Histona Desacetilases/química , Humanos , Modelos Moleculares , Conformação Proteica , Sirtuína 1/química , Ressonância de Plasmônio de SuperfícieRESUMO
Protein arginine methyltransferases (PRMTs) play important roles in several cellular processes, including signaling, gene regulation, and transport of proteins and nucleic acids, to impact growth, differentiation, proliferation, and development. PRMT5 symmetrically di-methylates the two-terminal ω-guanidino nitrogens of arginine residues on substrate proteins. PRMT5 acts as part of a multimeric complex in concert with a variety of partner proteins that regulate its function and specificity. A core component of these complexes is the WD40 protein MEP50/WDR77/p44, which mediates interactions with binding partners and substrates. We have determined the crystal structure of human PRMT5 in complex with MEP50 (methylosome protein 50), bound to an S-adenosylmethionine analog and a peptide substrate derived from histone H4. The structure of the surprising hetero-octameric complex reveals the close interaction between the seven-bladed ß-propeller MEP50 and the N-terminal domain of PRMT5, and delineates the structural elements of substrate recognition.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteína-Arginina N-Metiltransferases/química , Domínio Catalítico , Cristalografia por Raios X , Dimerização , Humanos , Modelos Moleculares , Conformação ProteicaRESUMO
The nuclear pore complex (NPC), embedded in the nuclear envelope, is a large, dynamic molecular assembly that facilitates exchange of macromolecules between the nucleus and the cytoplasm. The yeast NPC is an eightfold symmetric annular structure composed of ~456 polypeptide chains contributed by ~30 distinct proteins termed nucleoporins. Nup116, identified only in fungi, plays a central role in both protein import and mRNA export through the NPC. Nup116 is a modular protein with N-terminal "FG" repeats containing a Gle2p-binding sequence motif and a NPC targeting domain at its C-terminus. We report the crystal structure of the NPC targeting domain of Candida glabrata Nup116, consisting of residues 882-1034 [CgNup116(882-1034)], at 1.94 Å resolution. The X-ray structure of CgNup116(882-1034) is consistent with the molecular envelope determined in solution by small-angle X-ray scattering. Structural similarities of CgNup116(882-1034) with homologous domains from Saccharomyces cerevisiae Nup116, S. cerevisiae Nup145N, and human Nup98 are discussed.
Assuntos
Proteínas Fúngicas/química , Complexo de Proteínas Formadoras de Poros Nucleares/química , Poro Nuclear/química , Proteínas de Saccharomyces cerevisiae/química , Homologia de Sequência de Aminoácidos , Sequência de Aminoácidos , Candida glabrata/química , Cristalografia por Raios X , Humanos , Dados de Sequência Molecular , Complexos Multiproteicos/química , Membrana Nuclear/química , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/químicaRESUMO
Adenine deaminase (ADE) from the amidohydrolase superfamily (AHS) of enzymes catalyzes the conversion of adenine to hypoxanthine and ammonia. Enzyme isolated from Escherichia coli was largely inactive toward the deamination of adenine. Molecular weight determinations by mass spectrometry provided evidence that multiple histidine and methionine residues were oxygenated. When iron was sequestered with a metal chelator and the growth medium supplemented with Mn(2+) before induction, the post-translational modifications disappeared. Enzyme expressed and purified under these conditions was substantially more active for adenine deamination. Apo-enzyme was prepared and reconstituted with two equivalents of FeSO(4). Inductively coupled plasma mass spectrometry and Mössbauer spectroscopy demonstrated that this protein contained two high-spin ferrous ions per monomer of ADE. In addition to the adenine deaminase activity, [Fe(II) /Fe(II) ]-ADE catalyzed the conversion of H(2)O(2) to O(2) and H(2)O. The values of k(cat) and k(cat)/K(m) for the catalase activity are 200 s(-1) and 2.4 × 10(4) M(-1) s(-1), respectively. [Fe(II)/Fe(II)]-ADE underwent more than 100 turnovers with H(2)O(2) before the enzyme was inactivated due to oxygenation of histidine residues critical for metal binding. The iron in the inactive enzyme was high-spin ferric with g(ave) = 4.3 EPR signal and no evidence of anti-ferromagnetic spin-coupling. A model is proposed for the disproportionation of H(2)O(2) by [Fe(II)/Fe(II)]-ADE that involves the cycling of the binuclear metal center between the di-ferric and di-ferrous oxidation states. Oxygenation of active site residues occurs via release of hydroxyl radicals. These findings represent the first report of redox reaction catalysis by any member of the AHS.
Assuntos
Aminoidrolases/metabolismo , Catalase/metabolismo , Escherichia coli/enzimologia , Ferro/metabolismo , Aminoidrolases/química , Aminoidrolases/genética , Escherichia coli/química , Escherichia coli/genética , Peróxido de Hidrogênio/metabolismo , Radical Hidroxila/metabolismo , Ferro/química , Modelos Moleculares , Mutagênese , Oxirredução , Superóxidos/metabolismoAssuntos
Complexo de Proteínas Formadoras de Poros Nucleares/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Humanos , Antígenos de Histocompatibilidade Menor , Modelos Moleculares , Estrutura Terciária de Proteína , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
Per-Arnt-Sim (PAS) domain-containing protein kinase (PASK) is an evolutionary conserved protein kinase that coordinates cellular metabolism with metabolic demand in yeast and mammals. The molecular mechanisms underlying PASK regulation, however, remain unknown. Herein, we describe a crystal structure of the kinase domain of human PASK, which provides insights into the regulatory mechanisms governing catalysis. We show that the kinase domain adopts an active conformation and has catalytic activity in vivo and in vitro in the absence of activation loop phosphorylation. Using site-directed mutagenesis and structural comparison with active and inactive kinases, we identified several key structural features in PASK that enable activation loop phosphorylation-independent activity. Finally, we used combinatorial peptide library screening to determine that PASK prefers basic residues at the P-3 and P-5 positions in substrate peptides. Our results describe the key features of the PASK structure and how those features are important for PASK activity and substrate selection.
Assuntos
Proteínas Serina-Treonina Quinases/química , Ativação Enzimática/fisiologia , Humanos , Mutagênese Sítio-Dirigida , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Relação Estrutura-AtividadeRESUMO
A strategy for increasing the efficiency of protein crystallization/structure determination with mass spectrometry has been developed. This approach combines insights from limited proteolysis/mass spectrometry and crystallization via in situ proteolysis. The procedure seeks to identify protease-resistant polypeptide chain segments from purified proteins on the time-scale of crystal formation, and subsequently crystallizing the target protein in the presence of the optimal protease at the right relative concentration. We report our experience with 10 proteins of unknown structure, two of which yielded high-resolution X-ray structures. The advantage of this approach comes from its ability to select only those structure determination candidates that are likely to benefit from application of in situ proteolysis, using conditions most likely to result in formation of a stable proteolytic digestion product suitable for crystallization.
Assuntos
Espectrometria de Massas/métodos , Conformação Proteica , Proteínas , Cristalização/métodos , Cristalografia por Raios X , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Fragmentos de Peptídeos/química , Peptídeo Hidrolases/metabolismo , Proteínas/química , Proteínas/metabolismoAssuntos
Benzoatos/metabolismo , Canais Iônicos/química , Porinas/química , Pseudomonas fluorescens/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , Canais Iônicos/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Porinas/metabolismo , Conformação Proteica , Homologia de Sequência de AminoácidosAssuntos
Complexo de Proteínas Formadoras de Poros Nucleares/química , Processamento de Proteína Pós-Traducional , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Cromatografia em Gel , Cristalografia por Raios X , Humanos , Espectroscopia de Ressonância Magnética , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Homologia Estrutural de ProteínaRESUMO
PHR [PAM (protein associated with Myc)-HIW (Highwire)-RPM-1 (regulator of presynaptic morphology 1)] proteins are conserved, large multi-domain E3 ubiquitin ligases with modular architecture. PHR proteins presynaptically control synaptic growth and axon guidance and postsynaptically regulate endocytosis of glutamate receptors. Dysfunction of neuronal ubiquitin-mediated proteasomal degradation is implicated in various neurodegenerative diseases. PHR proteins are characterized by the presence of two PHR domains near the N-terminus, which are essential for proper localization and function. Structures of both the first and second PHR domains of Mus musculus (mouse) Phr1 (MYC binding protein 2, Mycbp2) have been determined, revealing a novel beta sandwich fold composed of 11 antiparallel beta-strands. Conserved loops decorate the apical side of the first PHR domain (MmPHR1), yielding a distinct conserved surface feature. The surface of the second PHR domain (MmPHR2), in contrast, lacks significant conservation. Importantly, the structure of MmPHR1 provides insights into a loss-of-function mutation, Gly1092-->Glu, observed in the Caenorhabditis elegans ortholog RPM-1.
Assuntos
Substituição de Aminoácidos/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Transporte/química , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Sequência de Aminoácidos , Animais , Caenorhabditis elegans , Cristalografia por Raios X , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Alinhamento de Sequência , Ubiquitina-Proteína LigasesRESUMO
Upon removal of the regulatory insert (RI), the first nucleotide binding domain (NBD1) of human cystic fibrosis transmembrane conductance regulator (CFTR) can be heterologously expressed and purified in a form that remains stable without solubilizing mutations, stabilizing agents or the regulatory extension (RE). This protein, NBD1 387-646(Delta405-436), crystallizes as a homodimer with a head-to-tail association equivalent to the active conformation observed for NBDs from symmetric ATP transporters. The 1.7-A resolution X-ray structure shows how ATP occupies the signature LSGGQ half-site in CFTR NBD1. The DeltaF508 version of this protein also crystallizes as a homodimer and differs from the wild-type structure only in the vicinity of the disease-causing F508 deletion. A slightly longer construct crystallizes as a monomer. Comparisons of the homodimer structure with this and previously published monomeric structures show that the main effect of ATP binding at the signature site is to order the residues immediately preceding the signature sequence, residues 542-547, in a conformation compatible with nucleotide binding. These residues likely interact with a transmembrane domain intracellular loop in the full-length CFTR channel. The experiments described here show that removing the RI from NBD1 converts it into a well-behaved protein amenable to biophysical studies yielding deeper insights into CFTR function.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/química , Modelos Moleculares , Conformação Proteica , Estrutura Terciária de Proteína/genética , Sítios de Ligação/genética , Clonagem Molecular , Cristalização , Regulador de Condutância Transmembrana em Fibrose Cística/isolamento & purificação , Primers do DNA/genética , Dimerização , Humanos , Mutação/genéticaRESUMO
The mechanistically diverse enolase superfamily is a paradigm for elucidating Nature's strategies for divergent evolution of enzyme function. Each of the different reactions catalyzed by members of the superfamily is initiated by abstraction of the alpha-proton of a carboxylate substrate that is coordinated to an essential Mg(2+). The muconate lactonizing enzyme (MLE) from Pseudomonas putida, a member of a family that catalyzes the syn-cycloisomerization of cis,cis-muconate to (4S)-muconolactone in the beta-ketoadipate pathway, has provided critical insights into the structural bases for evolution of function within the superfamily. A second, divergent family of homologous MLEs that catalyzes anti-cycloisomerization has been identified. Structures of members of both families liganded with the common (4S)-muconolactone product (syn, Pseudomonas fluorescens, gi 70731221 ; anti, Mycobacterium smegmatis, gi 118470554 ) document that the conserved Lys at the end of the second beta-strand in the (beta/alpha)(7)beta-barrel domain serves as the acid catalyst in both reactions. The different stereochemical courses (syn and anti) result from different structural strategies for determining substrate specificity: although the distal carboxylate group of the cis,cis-muconate substrate attacks the same face of the proximal double bond, opposite faces of the resulting enolate anion intermediate are presented to the conserved Lys acid catalyst. The discovery of two families of homologous, but stereochemically distinct, MLEs likely provides an example of "pseudoconvergent" evolution of the same function from different homologous progenitors within the enolase superfamily, in which different spatial arrangements of active site functional groups and substrate specificity determinants support catalysis of the same reaction.
Assuntos
Evolução Molecular , Liases Intramoleculares/metabolismo , Fosfopiruvato Hidratase/metabolismo , Biocatálise , Clonagem Molecular , Cristalografia por Raios X , Liases Intramoleculares/química , Liases Intramoleculares/genética , Modelos Moleculares , Mycobacterium smegmatis/enzimologia , Fosfopiruvato Hidratase/química , Fosfopiruvato Hidratase/genética , Filogenia , Conformação Proteica , Pseudomonas fluorescens/enzimologia , Pseudomonas putida/enzimologia , Estereoisomerismo , Especificidade por SubstratoRESUMO
Phase II of the Protein Structure Initiative, funded by the NIH NIGMS (National Institute of General Medical Sciences), is a 5-year effort to determine thousands of protein structures. The New York SGX Research Center for Structural Genomics (NYSGXRC) is one of the four large-scale production centers tasked with determining 100-200 structures annually. Almost all protein production is carried out using the high throughput structural biology platform at SGX Pharmaceuticals (SGX), which supplies 120 or more ultrapure proteins per month for NYSGXRC crystallization and structure determination activities. Protocols for PCR, cloning, expression/solubility testing, fermentation, purification, and crystallization are described. General protocols and detailed experimental results for each target are updated weekly at the public PepcDB website (pepcdb.pdb.org/), and all NYSGXRC clones should be available in 2008 through the PlasmID resource operated by the Harvard Institute of Proteomics.
Assuntos
Proteínas/química , Proteínas/isolamento & purificação , Proteômica/métodos , Proteômica/organização & administração , Clonagem Molecular/métodos , Cristalografia por Raios X/métodos , Cidade de Nova Iorque , Reação em Cadeia da Polimerase/métodos , Proteínas/genéticaRESUMO
The rate-limiting step in the transit of absorbed dietary fat across the enterocyte is the generation of the pre-chylomicron transport vesicle (PCTV) from the endoplasmic reticulum (ER). This vesicle does not require coatomer-II (COPII) proteins for budding from the ER membrane and contains vesicle-associated membrane protein 7, found in intestinal ER, which is a unique intracellular location for this SNARE protein. We wished to identify the protein(s) responsible for budding this vesicle from ER membranes in the absence of the requirement for COPII proteins. We chromatographed rat intestinal cytosol on Sephacryl S-100 and found that PCTV budding activity appeared in the low molecular weight fractions. Additional chromatographic steps produced a single major and several minor bands on SDS-PAGE. By tandem mass spectroscopy, the bands contained both liver and intestinal fatty acid-binding proteins (L- and I-FABP) as well as four other proteins. Recombinant proteins for each of the six proteins identified were tested for PCTV budding activity; only L-FABP and I-FABP (23% the activity of L-FABP) were active. The vesicles generated by L-FABP were sealed, contained apolipoproteins B48 and AIV, were of the same size as PCTV on Sepharose CL-6B, and by electron microscopy, excluded calnexin and calreticulin but did not fuse with cis-Golgi nor did L-FABP generate COPII-dependent vesicles. Gene-disrupted L-FABP mouse cytosol had 60% the activity of wild type mouse cytosol. We conclude that L-FABP can select cargo for and bud PCTV from intestinal ER membranes.