A method has been devised for the electro- MATERIALS AND METHODS Immunogens and Immunization Procedures. Escherichia coli ribosomal proteins L7 and L12 were extracted (10) from 50S subunits and purified as described (11) by ion-exchange chromatography on carboxymethyl-and DEAE-cellulose. Antibodies were raised in a goat by injecting 250 jig of protein emulsified with complete Freund's adjuvant intracutaneously distributed over several sites. Bacillus pertussis vaccine (1.5 ml of Bordet-Gengou vaccine, Schweizerisches Serum-und Impfinstitut, Bern, Switzerland) was given subcutaneously with every antigen injection. Booster injections of the same formulation were given on days 38, 79, and 110. The animal was bled on day 117.Subunits from chicken liver ribosomes (12) were combined in equimolar amounts, and 200-iAg aliquots were emulsified with 125 Al of complete Freund's adjuvant injected at one intraperitoneal and four subcutaneous sites into BALB/c mice. Booster injections of 400 ,ig of ribosomes in saline were given intraperitoneally on days 33, 57, 58, and 59. The animals were bled on day 71. Electrophoretic Blotting Procedures. Proteins were first subjected to electrophoresis in the presence of urea either in two dimensions (12) or in one-dimensional slab gels corresponding to the second dimension of the same two-dimensional system. The proteins were then transferred to nitrocellulose sheets as follows. The physical assembly used is shown diagrammatically in Fig. 1. A sheet of nitrocellulose (0.45 ,im pore size in roll form, Millipore) was briefly wetted with water and laid on a scouring pad (Scotch-Brite) which was supported by a stiff plastic grid (disposable micropipette tray, Medical Laboratory Automation, Inc., New York). The gel to be blotted was put on the nitrocellulose sheet and care was taken to remove all air bubbles. A second pad and plastic grid were added and rubber bands were strung around all layers. The gel was thus firmly and evenly pressed against the nitrocellulose sheet. The assembly was put into an electrophoretic destaining chamber with the nitrocellulose sheet facing the cathode. The chamber contained 0.7% acetic acid. A voltage gradient of 6 V/cm was applied for 1 hr.For polyacrylamide electrophoresis in the presence of sodium dodecyl sulfate (13) instead of urea, the procedure was as det To whom reprint requests should be addressed. 4350The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

No abstract

In protein interaction analysis, one promising method to identify the involved proteins and to characterize interacting sites at the same time is the mass spectrometric analysis of enzymatic hydrolysates of covalently cross-linked complexes. While protein identification can be accomplished by the methodology developed for proteome analysis, the unequivocal detection and characterization of cross-linked sites remained involved without selection criteria for linked peptides in addition to mass. To provide such criteria, we incorporated cross-links with a distinct isotope pattern into the microtubule-destabilizing protein Op18/stathmin (Op18) and into complexes formed by Op18 with tubulin. The deuterium-labeled cross-linking reagents bis(sulfosuccinimidyl)-glutarate-d4, -pimelate-d4, and -sebacate-d4 were prepared together with their undeuterated counterparts and applied as a 1:1 mixture of the respective d0 and d4 isotopomers. The resulting d0/d4 isotope tags allowed a straightforward mass spectrometric detection of peptides carrying the linker even in complex enzymatic protein hydrolysates. In the structure elucidation of the linked peptides by MS/MS, the assignment of the linked amino acids was again greatly facilitated by the d0/d4 tag. By applying two cross-linkers with similar reactivity but different spacer length in parallel, even doublets with very low intensity could be assigned with high confidence in MS and MS/MS spectra. Since in the Op18-tubulin complexes only a limited number of peptides carried the linker, the identification of the involved proteins per se was not impeded, thus accomplishing both protein identification and characterization of interacting sites in the same experiment. This novel methodology allowed us to significantly refine the current view of the complex between Op18 and tubulin corroborating the tubulin "capping" activity of the N-terminal domain of Op18.

Lin28 inhibits the biogenesis of let-7 miRNAs through a direct interaction with the terminal loop of pre-let-7. This interaction requires the zinc-knuckle domains of Lin28. We show that the zinc knuckle domains of Lin28 are sufficient to provide binding selectivity for pre-let-7 miRNAs and present the NMR structure of human Lin28 zinc knuckles bound to the short sequence 5'-AGGAGAU-3'. The structure reveals that each zinc knuckle recognizes an AG dinucleotide separated by a single nucleotide spacer. This defines a new 5'-NGNNG-3' consensus motif that explains how Lin28 selectively recognizes pre-let-7 family members. Binding assays in cell lysates and functional assays in cultured cells demonstrate that the interactions observed in the solution structure also occur between the full-length protein and members of the pre-let-7 family. The consensus sequence explains several seemingly disparate previously published observations on the binding properties of Lin28.

There is a pressing need for immunosuppressants with an improved safety profile. The search for novel approaches to blocking T-cell activation led to the development of the selective protein kinase C (PKC) inhibitor AEB071 (sotrastaurin). In cell-free kinase assays AEB071 inhibited PKC, with K i values in the subnanomolar to low nanomolar range. Upon T-cell stimulation, AEB071 markedly inhibited in situ PKC catalytic activity and selectively affected both the canonical nuclear factor-B and nuclear factor of activated T cells (but not activator protein-1) transactivation pathways. In primary human and mouse T cells, AEB071 treatment effectively abrogated at low nanomolar concentration markers of early T-cell activation, such as interleukin-2 secretion and CD25 expression. Accordingly, the CD3/CD28 antibody-and alloantigen-induced T-cell proliferation responses were potently inhibited by AEB071 in the absence of nonspecific antiproliferative effects. Unlike former PKC inhibitors, AEB071 did not enhance apoptosis of murine T-cell blasts in a model of activation-induced cell death. Furthermore, AEB071 markedly inhibited lymphocyte function-associated antigen-1-mediated T-cell adhesion at nanomolar concentrations. The mode of action of AEB071 is different from that of calcineurin inhibitors, and AEB071 and cyclosporine A seem to have complementary effects on T-cell signaling pathways.Phosphorylation of serine, threonine, and tyrosine residues is a primary mechanism for regulating protein function in eukaryotic cells. Protein kinases, the enzymes that catalyze these reactions, regulate essentially all cellular processes and have thus emerged as therapeutic targets for many human diseases. However, nearly all protein kinase inhibitors target the ATP binding site. For this reason, design of inhibitors that selectively target even a subset of the approximately 570 related human protein kinase domains continues to be a daunting challenge. Nevertheless, small-molecule inhibitors of Abelson tyrosine kinase and epidermal growth factor receptor have been recently developed into clinically useful anticancer drugs (for review, see Medinger and Drevs, 2005).The protein kinase C (PKC) family of serine/threonine kinases plays a central role in the adaptive immune system. PKC can be grouped into three categories according to the