The nuclear GYF protein CD2BP2/U5-52K is required for T cell homeostasis.

The question whether interference with the ubiquitous splicing machinery can lead to cell-type specific perturbation of cellular function is addressed here by T cell specific ablation of the general U5 snRNP assembly factor CD2BP2/U5-52K. This protein defines the family of nuclear GYF domain containing proteins that are ubiquitously expressed in eukaryotes with essential functions ascribed to early embryogenesis and organ function. Abrogating CD2BP2/U5-52K in T cells, allows us to delineate the consequences of splicing machinery interferences for T cell development and function. Increased T cell lymphopenia and T cell death are observed upon depletion of CD2BP2/U5-52K. A substantial increase in exon skipping coincides with the observed defect in the proliferation/differentiation balance in the absence of CD2BP2/U5-52K. Prominently, skipping of exon 7 in Mdm4 is observed, coinciding with upregulation of pro-apoptotic gene expression profiles upon CD2BP2/U5-52K depletion. Furthermore, we observe enhanced sensitivity of naïve T cells compared to memory T cells to changes in CD2BP2/U5-52K levels, indicating that depletion of this general splicing factor leads to modulation of T cell homeostasis. Given the recent structural characterization of the U5 snRNP and the crosslinking mass spectrometry data given here, design of inhibitors of the U5 snRNP conceivably offers new ways to manipulate T cell function in settings of disease.

CD4+ T Cell Epitope Identification from Complex Parasite Antigen Mixtures.

Antigen complexity represents a major challenge for scoring CD4+ T cell immunogenicity, a key hallmark of immunity and with great potential to improve vaccine development. In this chapter, we provide a comprehensive picture of a pipeline that can be applied to virtually any complex antigen to overcome different limitations. Antigens are characterized by Mass Spectrometry to determine the available protein sources and their abundances. A reconstituted in vitro antigen processing system is applied along with bioinformatics tools to prioritize the list of candidates. Finally, the immunogenicity of candidate peptides is validated ex vivo using PBMCs from HLA-typed individuals. This protocol compiles the essential information for executing the whole pipeline while focusing on the candidate epitope prioritizing scheme.

Target Recognition in Tandem WW Domains: Complex Structures for Parallel and Antiparallel Ligand Orientation in h-FBP21 Tandem WW.

Protein-protein interactions often rely on specialized recognition domains, such as WW domains, which bind to specific proline-rich sequences. The specificity of these protein-protein interactions can be increased by tandem repeats, i.e., two WW domains connected by a linker. With a flexible linker, the WW domains can move freely with respect to each other. Additionally, the tandem WW domains can bind in two different orientations to their target sequences. This makes the elucidation of complex structures of tandem WW domains extremely challenging. Here, we identify and characterize two complex structures of the tandem WW domain of human formin-binding protein 21 and a peptide sequence from its natural binding partner, the core-splicing protein SmB/B'. The two structures differ in the ligand orientation and, consequently, also in the relative orientation of the two WW domains. We analyze and probe the interactions in the complexes by molecular simulations and NMR experiments. The workflow to identify the complex structures uses molecular simulations, density-based clustering, and peptide docking. It is designed to systematically generate possible complex structures for repeats of recognition domains. These structures will help us to understand the synergistic and multivalency effects that generate the astonishing versatility and specificity of protein-protein interactions.

CD4+ Th immunogenicity of the Ascaris spp. secreted products.

Ascaris spp. is a major health problem of humans and animals alike, and understanding the immunogenicity of its antigens is required for developing urgently needed vaccines. The parasite-secreted products represent the most relevant, yet complex (>250 proteins) antigens of Ascaris spp. as defining the pathogen-host interplay. We applied an in vitro antigen processing system coupled to quantitative proteomics to identify potential CD4+ Th cell epitopes in Ascaris-secreted products. This approach considerably restricts the theoretical list of epitopes using conventional CD4+ Th cell epitope prediction tools. We demonstrate the specificity and utility of our approach on two sets of candidate lists, allowing us identifying hits excluded by either one or both computational methods. More importantly, one of the candidates identified experimentally, clearly demonstrates the presence of pathogen-reactive T cells in healthy human individuals against these antigens. Thus, our work pipeline identifies the first human T cell epitope against Ascaris spp. and represents an easily adaptable platform for characterization of complex antigens, in particular for those pathogens that are not easily amenable for in vivo experimental validation.

FBP21's C-Terminal Domain Remains Dynamic When Wrapped around the c-Sec63 Unit of Brr2 Helicase.

Based on our recent finding that FBP21 regulates human Brr2 helicase activity involved in the activation of the spliceosomal B-complex, we investigated the structural and dynamic contribution of FBP21 to the interaction. By using NMR spectroscopy, we could show that the 50 C-terminal residues of FBP21 (FBP21326-376), which are sufficient to fully form the interaction with the C-terminal Sec63 unit of Brr2 (Brr2C-Sec63), adopt a random-coil conformation in their unbound state. Upon interaction with Brr2C-Sec63, 42 residues of FBP21326-376 cover the large binding site on Brr2C-Sec63 in an extended conformation. Short charged motifs are steering complex formation, still allowing the bound state to retain dynamics. Based on fragment docking in combination with experimental restraints, we present models of the complex structure. The FBP21326-376/Brr2C-Sec63 interaction thus presents an example of an intrinsically disordered protein/ordered-protein interaction in which a large binding site provides high specificity and, in combination with conformational disorder, displays a relatively high affinity.

Exploring monovalent and multivalent peptides for the inhibition of FBP21-tWW.

The coupling of peptides to polyglycerol carriers represents an important route towards the multivalent display of protein ligands. In particular, the inhibition of low affinity intracellular protein-protein interactions can be addressed by this design. We have applied this strategy to develop binding partners for FBP21, a protein which is important for the splicing of pre-mRNA in the nucleus of eukaryotic cells. Firstly, by using phage display the optimized sequence WPPPPRVPR was derived which binds with K Ds of 80 µM and 150 µM to the individual WW domains and with a K D of 150 µM to the tandem-WW1-WW2 construct. Secondly, this sequence was coupled to a hyperbranched polyglycerol (hPG) that allowed for the multivalent display on the surface of the dendritic polymer. This novel multifunctional hPG-peptide conjugate displayed a K D of 17.6 µM which demonstrates that the new carrier provides a venue for the future inhibition of proline-rich sequence recognition by FBP21 during assembly of the spliceosome.

Custom-fit ruthenium(II) metallopeptides: a new twist to DNA binding with coordination compounds.

A new bipyridine building block has been used for the solid-phase synthesis of dinuclear DNA-binding ruthenium(II) metallopeptides. Detailed spectroscopic studies suggest that these compounds bind to the DNA by insertion into the DNA minor groove. Moreover, the potential of the solid-phase peptide synthesis approach is demonstrated by the straightforward synthesis of an octaarginine derivative that shows effective cellular internalization and cytotoxicity linked with strong DNA interaction, as evidenced by steady-state fluorescence spectroscopy and AFM studies.