Hfq, a protein required for small RNA (sRNA)-mediated regulation in bacteria, binds RNA with low-nanomolar K d values and long half-lives of complexes (>100 min). This cannot be reconciled with the 1-2-min response time of regulation in vivo. We show that RNAs displace each other on Hfq on a short time scale by RNA concentrationdriven (active) cycling. Already at submicromolar concentrations of competitor RNA, half-lives of RNA-Hfq complexes are »1 min. We propose that competitor RNA associates transiently with RNA-Hfq complexes, RNAs exchange binding sites, and one of the RNAs eventually dissociates. This solves the ''strong binding-high turnover'' paradox and permits efficient use of the Hfq pool. The homohexameric Hfq ring displays two faces: proximal and distal. Hfq-RNA interactions show a preference of U-rich for proximal and A-rich RNA sequences for distal face binding (de Haseth and Uhlenbeck 1980a;Mikulecky et al. 2004). Simultaneous binding may occur on both sides as well, which could facilitate intermolecular base-pairing and regulation (Rajkowitsch and Schroeder 2007).Structures of Hfq from E. coli, Staphylococcus aureus, and Pseudomonas aeroginosa have been determined by X-ray crystallography (Schumacher et al. 2002;Sauter et al. 2003;Nikulin et al. 2005). Two cocrystal structures support two distinct binding surfaces: In S. aureus Hfq, AU 5 G RNA is bound around the inner rim of the proximal face (Schumacher et al. 2002), and E. coli Hfq has oligo-A bound on the distal face (Link et al. 2009 Holmqvist et al. 2010). Thus, if binding-competent RNAs were in molar excess, almost all Hfq would be bound to RNAs. Hfq-RNA dissociation rate constants in vitro are too low to be compatible with a biologically relevant time scale; half-lives of complexes are in the range of a generation time. If newly induced sRNAs only could access free Hfq after its dissociation from bound RNAs, their activity should be severely delayed. Yet, the time frame from induction of an sRNA to a significant regulatory effect is short (1-2 min) (Massé et al. 2003), and hence sRNAs can acquire Hfq rapidly. This highlights a paradox, with Hfq being tightly sequestered by the intracellular pool of RNAs, contrasted by the need of new sRNAs to rapidly access Hfq. We considered here a conventional cycling model (dissociative/passive) (Fig. 1A) and associative/active cycling (Fig. 1B). In model A, newly synthesized RNA (Fig. 1A, in red) can only bind Hfq after the resident RNA (Fig. 1A, in blue) has dissociated; i.e., the rate of binding of the incoming RNA is limited by the Hfq-RNA dissociation rate constant and is not affected by the concentration of the free RNA. In model B, free RNA transiently binds the Hfq-RNA complex, whereupon one of the RNAs eventually dissociates. Thus, the dissociation rate of the bound RNA is a function of the concentration of the free RNA (Fig. 1B). This would render cycling much more rapidly, and the intracellular pool of binder RNAs would rapidly equilibrate on Hfq. The two models are distinguishable, since th...

A-kinase anchoring proteins (AKAPs) tether protein kinase A (PKA) and other signaling proteins to defined intracellular sites, thereby establishing compartmentalized cAMP signaling. AKAP-PKA interactions play key roles in various cellular processes, including the regulation of cardiac myocyte contractility. We discovered small molecules, 3,3′-diamino-4,4′-dihydroxydiphenylmethane (FMP-API-1) and its derivatives, which inhibit AKAP-PKA interactions in vitro and in cultured cardiac myocytes. The molecules bind to an allosteric site of regulatory subunits of PKA identifying a hitherto unrecognized region that controls AKAP-PKA interactions. FMP-API-1 also activates PKA. The net effect of FMP-API-1 is a selective interference with compartmentalized cAMP signaling. In cardiac myocytes, FMP-API-1 reveals a novel mechanism involved in terminating β-adrenoreceptor-induced cAMP synthesis. In addition, FMP-API-1 leads to an increase in contractility of cultured rat cardiac myocytes and intact hearts. Thus, FMP-API-1 represents not only a novel means to study compartmentalized cAMP/PKA signaling but, due to its effects on cardiac myocytes and intact hearts, provides the basis for a new concept in the treatment of chronic heart failure.

V-raf-1 murine leukemia viral oncogene homolog 1 (Raf-1) is a key activator of the ERK pathway and is a target for cross-regulation of this pathway by the cAMP signaling system. The cAMP-activated protein kinase, PKA, inhibits Raf-1 by phosphorylation on S259. Here, we show that the cAMP-degrading phosphodiesterase-8A (PDE8A) associates with Raf-1 to protect it from inhibitory phosphorylation by PKA, thereby enhancing Raf-1's ability to stimulate ERK signaling. PDE8A binds to Raf-1 with high (picomolar) affinity. Mapping of the interaction domain on PDE8A using peptide array technology identified amino acids 454-465 as the main binding site, which could be disrupted by mutation. A cell-permeable peptide corresponding to this region disrupted the PDE8A/Raf-1 interaction in cells, thereby reducing ERK activation and the cellular response to EGF. Overexpression of a catalytically inactive PDE8A in cells displayed a dominant negative phenotype on ERK activation. These effects were recapitulated at the organism level in genetically modified (PDE8A −/− ) mice. Similarly, PDE8 deletion in Drosophila melanogaster reduced basal ERK activation and sensitized flies to stress-induced death. We propose that PDE8A is a physiological regulator of Raf-1 signaling in some cells.

Cytokines of the interleukin (IL)-1 family regulate immune and inflammatory responses. The recently discovered IL-36 family members are involved in psoriasis, rheumatoid arthritis, and pulmonary diseases. Here, we show that IL-36α interacts with heme thereby contributing to its regulation. Based on in-depth spectroscopic analyses, we describe two heme-binding sites in IL-36α that associate with heme in a pentacoordinated fashion. Solution NMR analysis reveals structural features of IL-36α and its complex with heme. Structural investigation of a truncated IL-36α supports the notion that the N-terminus is necessary for association with its cognate receptor. Consistent with our structural studies, IL-36-mediated signal transduction was negatively regulated by heme in synovial fibroblast-like synoviocytes from rheumatoid arthritis patients. Taken together, our results provide a structural framework for heme-binding proteins and add IL-1 cytokines to the group of potentially heme-regulated proteins.

In an effort to gain further insight into the conformational and topographical requirements for recognition by the N-terminal SH2 domain of protein tyrosine phosphatase SHP-1, we synthesized a series of linear and cyclic peptides derived from the sequence surrounding phosphotyrosine 2267 in the receptor tyrosine kinase Ros (EGLNpYMVL). A molecular modeling approach was used to suggest peptide modifications sterically compatible with the N-SH2-peptide binding groove and possibly enhanced binding affinities compared to the parent peptide. The potencies of the synthesized compounds were evaluated by assaying their ability to stimulate phosphatase activity as well as by their binding affinities to the GST-fused N-SH2 domain of SHP-1. In the series of linear peptides, structural modifications of Ros pY2267 in positions pY + 1 to pY + 3 by amino acid residues structurally related to Phe, for example l-erythro/threo-Abu(betaPh) (5a, 5b), yielded ligands with increased binding affinity. The incorporation of d-amino acid residues at pY + 1 and pY + 3 led to inactive peptides. The replacement of Phe in both pY + 1 and pY + 3 by Tic (1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) was also not tolerated due to steric hindrance. Cyclic peptides (13, 14) that were linked via residues in positions pY - 1 (Lys) and pY + 2 (Asp/Glu) and contained a Gly residue in the bridging unit displayed much lower potencies for the stimulation of SHP-1 activity but increased binding affinities compared to Ros pY2267. They partially competed with Ros pY2267 in the activation assay. Such cyclic structures may serve as scaffolds for competitive SHP-1 inhibitor design targeting N-SH2 domain-protein interactions that block SHP-1 activation.