RESUMO
A novel type of isobaric internal peptide standard for quantitative proteomics is described. The standard is a synthetic peptide derived from the target peptide by positional permutation of two amino acids. This type of internal standard is denominated minimally permutated peptide analog (MIPA). MIPA can be differentiated from their target analytes by LC-MS due to individual retention times and/or by MS/MS due to specific fragment ions. Both quantification methods are demonstrated using peptide mixtures of low and high complexity.
Assuntos
Peptídeos/química , Proteômica/métodos , Sequência de Aminoácidos , Escherichia coli/genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Proteínas/análise , Proteínas/química , Proteínas/genética , Proteômica/normas , Padrões de Referência , Espectrometria de Massas em TandemRESUMO
Members of the transforming growth factor-beta/bone morphogenetic protein (TGF-beta/BMP) family of cytokines play crucial roles in animal development and are candidate molecules for host-parasite cross-communication in helminth diseases. TGF-beta/BMP-signalling involves binding of the cytokines to receptor kinases which subsequently activate intracellular transcription factors of the Smad family. We have previously characterized two members of the receptor-regulated Smad (R-Smad) family, EmSmadA and EmSmadB, from the human parasitic cestode Echinococcus multilocularis and now present evidence for two additional Smads that are expressed by the larval stages of the parasite. The full-length cDNAs coding for a third R-Smad, EmSmadC, and a common mediator Smad (Co-Smad), EmSmadD, were characterized. While EmSmadD displayed a typical Co-Smad structure, EmSmadC lacked the N-terminal MH1 domain which is typically found in Smads. In yeast two-hybrid analyses, EmSmadC and EmSmadD were capable of homo- and heterodimer formation with other Echinococcus Smads. Furthermore, EmSmadC displayed autonomous transcription activation activity and interacted with EmSkip, a member of the SNW/SKIP family of transcriptional regulators. In a heterologous expression system, EmSmadC was specifically phosphorylated by mammalian TGF-beta receptors, indicating that it is a member of the AR-Smad sub-family. Finally, in activity assays, the parasite's Erk-like kinase EmMPK1 phosphorylated EmSmadD, indicating cross-regulation between mitogen-activated protein kinase cascade- and TGF-beta/BMP-signalling in Echinococcus. The data presented herein significantly broaden our knowledge of Smad-signalling factors in E. multilocularis and will facilitate studies on TGF-beta/BMP-regulated genes in the parasite as well as TGF-beta/BMP mediated host-parasite cross-interaction during alveolar echinococcosis.
Assuntos
Echinococcus multilocularis/genética , Genes de Helmintos , Proteínas Smad/genética , Proteína Smad4/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas Morfogenéticas Ósseas/metabolismo , Clonagem Molecular , Equinococose/metabolismo , Interações Hospedeiro-Parasita , Humanos , Larva , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Alinhamento de Sequência , Proteínas Smad/metabolismo , Proteína Smad4/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
The shikimate pathway provides basic building blocks for a variety of aromatic compounds including aromatic amino acids, ubiquinone, folate and compounds of the secondary metabolism. The seven enzymatic reactions of the pathway lead to the generation of chorismate from simple products of the carbohydrate metabolism, namely erythrose 4-phosphate and phosphoenolpyruvate. The shikimate pathway is present in plants, bacteria, fungi and chromalveolata to which the apicomplexan parasites belong. As it is absent from humans, the enzymes of the shikimate pathway are attractive targets for antimicrobial drug development. Inhibition of the pathway is effective in controlling growth of certain apicomplexan parasites including the malaria parasite Plasmodium falciparum. Yet, despite being an attractive drug target, our knowledge of the shikimate pathway in this parasite group is lacking. The current review summarizes the available information and discusses aspects of the genetic organization of the shikimate pathway in apicomplexan parasites. Compounds acting on shikimate pathway enzymes will be presented and discussed in light of their impact for antiapicomplexan/antiplasmodial drug development.
Assuntos
Apicomplexa/metabolismo , Ácido Chiquímico/metabolismo , Animais , Apicomplexa/efeitos dos fármacos , Desenho de FármacosRESUMO
Biosynthesis of vitamins is fundamental to malaria parasites. Plasmodia synthesize the active form of vitamin B(6) (pyridoxal 5'-phosphate, PLP) using a PLP synthase complex. The EM analysis shown here reveals a random association pattern of up to 12 Pdx2 glutaminase subunits to the dodecameric Pdx1 core complex. Interestingly, Plasmodium falciparum PLP synthase organizes in fibers. The crystal structure shows differences in complex formation to bacterial orthologs as interface variations. Alternative positioning of an α helix distinguishes an open conformation from a closed state when the enzyme binds substrate. The pentose substrate is covalently attached through its C1 and forms a Schiff base with Lys84. Ammonia transfer between Pdx2 glutaminase and Pdx1 active sites is regulated by a transient tunnel. The mutagenesis analysis allows defining the requirement for conservation of critical methionines, whereas there is also plasticity in ammonia tunnel construction as seen from comparison across different species.
Assuntos
Glutaminase/química , Modelos Moleculares , Complexos Multiproteicos/metabolismo , Plasmodium falciparum/enzimologia , Conformação Proteica , Fosfato de Piridoxal/biossíntese , Amônia/metabolismo , Domínio Catalítico/genética , Cristalografia , Ativação Enzimática/genética , Glutaminase/metabolismo , Metionina/metabolismoRESUMO
Most organisms synthesise the B(6) vitamer pyridoxal 5-phosphate (PLP) via the glutamine amidotransferase PLP synthase, a large enzyme complex of 12 Pdx1 synthase subunits with up to 12 Pdx2 glutaminase subunits attached. Deletion analysis revealed that the C-terminus has four distinct functionalities: assembly of the Pdx1 monomers, binding of the pentose substrate (ribose 5-phosphate), formation of the reaction intermediate I(320), and finally PLP synthesis. Deletions of distinct C-terminal regions distinguish between these individual functions. PLP formation is the only function that is conferred to the enzyme by the C-terminus acting in trans, explaining the cooperative nature of the complex.