RESUMO
The tumor maintenance protein Tsg101 has recently gained much attention because of its involvement in endosomal sorting, virus release, cytokinesis, and cancerogenesis. The ubiquitin-E2-like variant (UEV) domain of the protein interacts with proline-rich sequences of target proteins that contain P(S/T)AP amino acid motifs and weakly binds to the ubiquitin moiety of proteins committed to sorting or degradation. Here we performed peptide spot analysis and phage display to refine the peptide binding specificity of the Tsg101 UEV domain. A mass spectrometric proteomics approach that combines domain-based pulldown experiments, binding site inactivation, and stable isotope labeling by amino acids in cell culture (SILAC) was then used to delineate the relative importance of the peptide and ubiquitin binding sites. Clearly "PTAP" interactions dominate target recognition, and we identified several novel binders as for example the poly(A)-binding protein 1 (PABP1), Sec24b, NFkappaB2, and eIF4b. For PABP1 and eIF4b the interactions were confirmed in the context of the corresponding full-length proteins in cellular lysates. Therefore, our results strongly suggest additional roles of Tsg101 in cellular regulation of mRNA translation. Regulation of Tsg101 itself by the ubiquitin ligase TAL (Tsg101-associated ligase) is most likely conferred by a single PSAP binding motif that enables the interaction with Tsg101 UEV. Together with the results from the accompanying article (Kofler, M., Schuemann, M., Merz, C., Kosslick, D., Schlundt, A., Tannert, A., Schaefer, M., Lührmann, R., Krause, E., and Freund, C. (2009) Proline-rich sequence recognition: I. Marking GYF and WW domain assembly sites in early spliceosomal complexes. Mol. Cell. Proteomics 8, 2461-2473) on GYF and WW domain pathways our work defines major proline-rich sequence-mediated interaction networks that contribute to the modular assembly of physiologically relevant protein complexes.
Assuntos
Proteínas de Ligação a DNA/química , Complexos Endossomais de Distribuição Requeridos para Transporte/química , Prolina/química , Proteômica/métodos , Fatores de Transcrição/química , Enzimas de Conjugação de Ubiquitina/química , Motivos de Aminoácidos , Sítios de Ligação , Clonagem Molecular , Células HeLa , Humanos , Espectrometria de Massas/métodos , Biblioteca de Peptídeos , Estrutura Terciária de Proteína , Proteoma , RNA Mensageiro/metabolismoRESUMO
Proline-rich sequences (PRS) and their recognition domains have emerged as transposable protein interaction modules during eukaryotic evolution. They are especially abundant in proteins associated with pre-mRNA splicing and likely assist in the formation of the spliceosome by binding to GYF and WW domains. Here we profile PRS-mediated interactions of the CD2BP2/52K GYF domain by a site-specific peptide inhibitor and stable isotope labeling/mass spectrometry analysis. Several PRS hubs with multiple proline-rich motifs exist that can recruit GYF and/or WW domains. Saturating the PRS sites by an isolated GYF domain inhibited splicing at the level of A complex formation. The interactions mediated by PRS are therefore important to the early phases of spliceosomal assembly.
Assuntos
Prolina/química , Estrutura Terciária de Proteína , Spliceossomos/metabolismo , Sítios de Ligação , Catálise , Linhagem Celular , Clonagem Molecular , Transferência Ressonante de Energia de Fluorescência , Humanos , Espectroscopia de Ressonância Magnética , Espectrometria de Massas/métodos , Peptídeos/química , Splicing de RNA , RNA Mensageiro/metabolismoRESUMO
A polymer support for the solid-phase synthesis of C-terminally labeled carboxylic acids has been developed. Fluorophore-labeled peptides were constructed directly on the amino group of resin-bound aminofluorescein. Fmoc-protected aminofluorescein was coupled onto tritylpolystyrene, and the free phenolic hydroxyl positions of the fluorescein were blocked with suitable protecting groups. The mode of attachment was analyzed and found to be selective for the phenoxy ether linkage. The conditions for peptide synthesis on the labeling resin were investigated, and a small library of C-terminally labeled peptides was prepared. The fluorescence quantum yields of C-terminally labeled peptides were determined and indicated the suitability of the compounds for imaging and binding experiments. The obtained peptides were therefore investigated as fluorescence polarization probes. Two different proline-rich binding domains of the GYF family-CD2BP2 and PERQ2-were targeted by peptides labeled either C- or N-terminally. Reversible binding constants were determined by fluorescence polarization measurements and were verified by competition experiments with the corresponding unlabeled peptide. As a second control, the binding constants were measured by NMR titration experiments, recording the HSQC NMR spectra of (15)N-labeled proteins in the presence of the peptide polarization probes. Ligands with higher affinities than all others known previously were identified for both GYF domains. The competition assay with the developed fluorescent probe has a high statistical reliability and can thus be used for screening of GYF domain inhibitors.
Assuntos
Aminas/química , Fluoresceínas/química , Sondas Moleculares/química , Peptídeos/química , Sequência de Aminoácidos , Ligação Competitiva , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Biblioteca de Peptídeos , Peptídeos/genética , Prolina/química , Espectrofotometria InfravermelhoRESUMO
Reversible oxidation of amino acids within intracellular proteins leads to local and/or global conformational changes in protein structure. Thus, the enzymatic activity or binding properties of a protein might be regulated by local changes in a cell's redox potential, mediated by the availability of reducing/oxidizing equivalents. Whereas it is well established that intracellular pools of oxidizable groups compensate for oxidative stress, far less is known about the molecular mechanisms that accompany transient and reversible oxidation of cytoplasmic proteins. Therefore, the intrinsic redox properties of proteins amenable to reversible oxidation need to be determined. Here we describe the application of NMR spectroscopy to derive the redox properties of intracellular proteins. As exemplified for thioredoxin 1, the Tnk-1 kinase SH3 domain, and the hSH3(N) domain of the T cell protein ADAP, the conformational changes associated with disulfide bond formation can be followed directly upon titration with different ratios of reduced to oxidized glutathione. Redox potentials can be measured accurately in homogeneous solutions and define the conditions under which regulatory oxidation of the respective protein may occur in the living cell.
Assuntos
Cisteína/química , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/química , Cisteína/metabolismo , Glutationa/química , Glutationa/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Modelos Moleculares , Oxirredução , Ligação Proteica , Proteínas/metabolismo , Tiorredoxinas/química , Tiorredoxinas/metabolismo , Domínios de Homologia de srcRESUMO
The yeast suppressor of myosin 2 protein (Smy2) interacts with mRNA-processing proteins through recognition of proline-rich sequences (PRS). Here, we describe the crystal structure of the GYF domain of Smy2 in association with a PRS from the yeast branch point binding protein (BBP/ScSF1). Complex formation requires that the beta-hairpin of the central PPGL motif of the ligand is accommodated by an extended hydrophobic cleft in the domain-a specificity feature that is maintained in the human protein GIGYF2. SILAC/MS experiments in combination with PRS site inhibition show that Smy2 associates with the Ccr4-NOT deadenylase complex, whereas GIGYF2 interacts not only with mRNA surveillance factors, but also with vesicular transport proteins and Atrophin-1. GIGYF2 is shown to associate with COPII-vesicle proteins and localize to the ER and Golgi in resting cells, whereas environmental challenge drives GIGYF2 into stress granules. The current study highlights the structural basis for PRS recognition by Smy2-type GYF domains, and implicates Smy2 and GIGYF2 in both mRNA processing and the secretory pathway.