Protein-protein interactions are often studied by chemical shift mapping using solution NMR spectroscopy. When heteronuclear data are available the interaction interface is usually predicted by combining the chemical shift changes of different nuclei to a single quantity, the combined chemical shift perturbation Deltadelta comb In this paper different procedures (published and non-published) to calculate Deltadelta comb are examined that include a variety of different functional forms and weighting factors for each nucleus. The predictive power of all shift mapping methods depends on the magnitude of the overlap of the chemical shift distributions of interacting and non-interacting residues and the cut-off criterion used. In general, the quality of the prediction on the basis of chemical shift changes alone is rather unsatisfactory but the combination of chemical shift changes on the basis of the Hamming or the Euclidian distance can improve the result. The corrected standard deviation to zero of the combined chemical shift changes can provide a reasonable cut-off criterion. As we show combined chemical shifts can also be applied for a more reliable quantitative evaluation of titration data.

So far, the annotation of translation initiation sites (TISs) has been based mostly upon bioinformatics rather than experimental evidence. We adapted ribosomal footprinting to puromycin-treated cells to generate a transcriptome-wide map of TISs in a human monocytic cell line. A neural network was trained on the ribosomal footprints observed at previously annotated AUG translation initiation codons (TICs), and used for the ab initio prediction of TISs in 5062 transcripts with sufficient sequence coverage. Functional interpretation suggested 2994 novel upstream open reading frames (uORFs) in the 5′ UTR, 1406 uORFs overlapping with the coding sequence, and 546 N-terminal protein extensions. The TIS detection method was validated on the basis of previously published alternative TISs and uORFs. Among primates, TICs in newly annotated TISs were significantly more conserved than control codons, both for AUGs and near-cognate codons. The transcriptome-wide map of novel candidate TISs derived as part of the study will shed further light on the way in which human proteome diversity is influenced by alternative translation initiation and regulation.

The PKD1 and PKD2 genes are the genes that are mutated in patients suffering from autosomal dominant polycystic kidney disease. The human PKD2 gene codes for a 968-amino acid long membrane protein called polycystin-2 that represents a cation channel whose activity can be regulated by Ca 2؉ ions. By CD, fluorescence, and NMR spectroscopy, we have studied a 117-amino acid-long fragment of the cytoplasmic domain of polycystin-2, polycystin-2-(680 -796) that was proposed to contain a Ca The PKD2 gene is one of the two genes mutated in patients with autosomal-dominant polycystic kidney disease (1). It encodes polycystin-2, a 968-amino acid-long protein with 6 putative membrane-spanning domains. According to structural predictions, both its N and C termini extend into the cytoplasm; furthermore, a pore-forming region has been postulated between the fifth and sixth membrane-spanning domains. By sequence comparison a coiled-coil domain and a Ca 2ϩ -binding EF-hand have been predicted in the C terminus of polycystin-2 (1) and a homology model has been published recently (2). However, so far only indirect evidence for the presence of these motifs has been presented. Electrophysiological measurements by several groups have demonstrated that polycystin-2 is a non-selective cation channel and that its channel activity is regulated by calcium. One report has shown the activation of polycystin-2 by the addition of 1 M Ca 2ϩ to the bath solution and its inhibition by millimolar concentrations of Ca 2ϩ (3). An R742X mutant protein, however, did not respond to the addition of Ca 2ϩ . These initial observations were confirmed by other publications that reported that Ca 2ϩ concentrations of up to 1.26 mM increase the probability for the open state of full-length polycystin-2 (4), whereas higher concentrations are inhibitory (4, 5). Again, the activity of a truncated protein, this time a 703-amino acid mutant, is not modulated by calcium (4).From the above it seems obvious that the C terminus via binding of Ca 2ϩ exerts a modulatory activity on the channel activity of polycystin-2. This part of the protein, however, also is of particular interest because it interacts with a wide variety of other proteins, among them polycystin-1, ion channels (inositol 1,4,5-trisphosphate receptor, polycystin-2 itself, and TRPC1), cytoskeletal proteins (␣-actinin, CD2AP, mDia1, troponin I, and tropomyosin-1), intracellular trafficking proteins (PACS-1, PACS-2, and PIGEA-14) and even a transcription factor (Id2) (6). Whether all of these interactions are direct or require additional cofactors is not clear at present; furthermore, it is not known whether any of them are cooperative or exclusive. We have therefore decided to subject the C terminus of polycystin-2 to an extensive biochemical and structural analysis. 3 The abbreviations used are: DSS, 2,2-dimethyl-2-silapentane-5-sulfonic acid; CDPK-␣, calmodulin-like domain of the calcium dependent soybean protein kinase ␣; HSQC, heteronuclear single quantum coherence; ISIC, intelligent structural infor...

Objective The ubiquitously expressed intracellular protein formerly designated p68 has been identified as autoantigen at both the antibody and the T cell level in rheumatoid arthritis (RA). Methods We used 2 independent approaches, Edman degradation and matrix‐assisted laser desorption ionization–time‐of‐flight mass spectrometry, to characterize p68, and we compared its features with those of the endoplasmic reticulum stress protein BiP. Results In synovial sections from RA patients, BiP was highly overexpressed as compared with control sections. Under in vitro stress conditions, BiP was found to translocate to the nucleus and the cell surface. BiP‐specific autoantibodies were present in 63% of 400 RA patients, in 7% of 200 patients with other rheumatic diseases, and in none of the healthy subjects. Thus, BiP‐specific autoantibodies represent a new diagnostic marker in RA. Furthermore, we found that BiP‐specific T cell reactivity was altered in RA. In healthy individuals and patients with other rheumatic diseases, BiP‐reactive T cells were undetectable. In RA, overt T cell reactivity to BiP was observed or could be induced by specifically blocking antigen presentation to potentially regulatory T cells. Conclusion Since overexpression of BiP has been shown to decrease the sensitivity of cells to killing by cytotoxic T cells, BiP overexpression and BiP‐specific autoimmunity may be involved in the pathogenesis of RA.

Thousands of tandem alternative splice sites (TASS) give rise to mRNA insertion/deletion variants with small size differences. Recent work has concentrated on the question of biological relevance in general, and the physiological regulation of TASS in particular. We have quantitatively studied 11 representative TASS cases in comparison to one mutually exclusive exon case and two cassette exons (CEs) using a panel of human and mouse tissues, as well as cultured cell lines. Tissues show small but significant differences in TASS isoform ratios, with a variance 4- to 20-fold lower than seen for CEs. Remarkably, in cultured cells, all studied alternative splicing (AS) cases showed a cell-density-dependent shift of isoform ratios with similar time series profiles. A respective genome-wide co-regulation of TASS splicing was shown by next-generation mRNA sequencing data. Moreover, data from human and mouse organs indicate that this co-regulation of TASS occurs in vivo, with brain showing the strongest difference to other organs. Together, the results indicate a physiological AS regulation mechanism that functions almost independently from the splice site context and sequence.