RESUMO
The coordinated breakdown of intracellular triglyceride (TG) stores requires the exquisitely regulated interaction of lipolytic enzymes with regulatory, accessory, and scaffolding proteins. Together they form a dynamic multiprotein network designated as the "lipolysome." Adipose triglyceride lipase (Atgl) catalyzes the initiating step of TG hydrolysis and requires comparative gene identification-58 (Cgi-58) as a potent activator of enzyme activity. Here, we identify adipocyte-type fatty acid-binding protein (A-Fabp) and other members of the fatty acid-binding protein (Fabp) family as interaction partners of Cgi-58. Co-immunoprecipitation, microscale thermophoresis, and solid phase assays proved direct protein/protein interaction between A-Fabp and Cgi-58. Using nuclear magnetic resonance titration experiments and site-directed mutagenesis, we located a potential contact region on A-Fabp. In functional terms, A-Fabp stimulates Atgl-catalyzed TG hydrolysis in a Cgi-58-dependent manner. Additionally, transcriptional transactivation assays with a luciferase reporter system revealed that Fabps enhance the ability of Atgl/Cgi-58-mediated lipolysis to induce the activity of peroxisome proliferator-activated receptors. Our studies identify Fabps as crucial structural and functional components of the lipolysome.
Assuntos
1-Acilglicerol-3-Fosfato O-Aciltransferase/metabolismo , Proteínas de Ligação a Ácido Graxo/metabolismo , Lipase/metabolismo , Complexos Multiproteicos/metabolismo , Triglicerídeos/metabolismo , 1-Acilglicerol-3-Fosfato O-Aciltransferase/genética , Tecido Adiposo/metabolismo , Animais , Células COS , Chlorocebus aethiops , Proteínas de Ligação a Ácido Graxo/genética , Humanos , Ligantes , Lipase/genética , Lipólise/genética , Lipossomos/metabolismo , Camundongos , Complexos Multiproteicos/genética , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , ProteóliseRESUMO
The universal second messenger cyclic di-GMP (cdG) is involved in the regulation of a diverse range of cellular processes in bacteria. The intracellular concentration of the dinucleotide is determined by the opposing actions of diguanylate cyclases and cdG-specific phosphodiesterases (PDEs). Whereas most PDEs have accessory domains that are involved in the regulation of their activity, the regulatory mechanism of this class of enzymes has remained unclear. Here, we use biophysical and functional analyses to show that the isolated EAL domain of a PDE from Escherichia coli (YahA) is in a fast thermodynamic monomer-dimer equilibrium, and that the domain is active only in its dimeric state. Furthermore, our data indicate thermodynamic coupling between substrate binding and EAL dimerization with the dimerization affinity being increased about 100-fold upon substrate binding. Crystal structures of the YahA-EAL domain determined under various conditions (apo, Mg(2+), cdG·Ca(2+) complex) confirm structural coupling between the dimer interface and the catalytic center. The built-in regulatory properties of the EAL domain probably facilitate its modular, functional combination with the diverse repertoire of accessory domains.
Assuntos
3',5'-GMP Cíclico Fosfodiesterases/metabolismo , GMP Cíclico/análogos & derivados , Proteínas de Escherichia coli/metabolismo , Sistemas do Segundo Mensageiro , 3',5'-GMP Cíclico Fosfodiesterases/química , 3',5'-GMP Cíclico Fosfodiesterases/genética , Sequência de Aminoácidos , Catálise , Domínio Catalítico , Cristalografia por Raios X , GMP Cíclico/química , GMP Cíclico/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Hidrólise , Dados de Sequência Molecular , Multimerização Proteica , Estrutura Terciária de ProteínaRESUMO
We present a theoretical and numerical analysis of the vibrational coupling between isotope-edited amino acids in protein dimers. Depending on the presence and magnitude of coupling between 13Calpha=O peptide bond oscillators, characteristic level splittings of vibrational eigenstates are predicted. For the example of the Gramicidin A ion channel polypeptide, we observe typical IR fingerprints for the head-to-head and the antiparallel double-helical conformation of the dimer. We suggest that these findings can be used to clearly identify the structure of polypeptide aggregates using a particularly simple isotope substitution pattern.
Assuntos
Algoritmos , Isótopos de Carbono/química , Oxigênio/química , Proteínas/química , Dimerização , Gramicidina/química , Isótopos , Modelos Moleculares , Espectroscopia de Luz Próxima ao InfravermelhoRESUMO
In this work, we present a simple kinetic model of horizontal gene transfer. It describes the processes of gene duplication, mutation, gene transfer and the regulation of the total size of the genome for genetically homogeneous prokaryotic species or strains. The emerging nonlinear system of first-order differential equations can be linearized at the stationary point. For selected models, we give an analytical solution for the number of foreign and native genes within a species. We identify a regime characterized by a fast gene transfer rate and species with a mixed genome, a slow gene transfer regime with pure organisms, and a crossover region. The data are compared to experiments, and the biological implications of our model are discussed.