Myeloid differentiation factor 88 (MyD88) plays a crucial role in the signaling pathways triggered by interleukin (IL)-1 and Toll-like receptors in several steps of innate host defense. A crucial event in this signaling pathway is represented by dimerization of MyD88, which allows the recruitment of downstream kinases like IRAK-1 and IRAK-4. Herein, we have investigated the function of the Toll/IL-1 receptor (TIR) domain in MyD88 homodimerization in cell-free and in vitro experimental settings by using epta-peptides that mimic the BB-loop region of the conserved TIR domain of different proteins. By using a pull-down assay with purified glutathione S-transferase-MyD88 TIR or co-immunoprecipitation experiments, we found that epta-peptides derived from the TIR domain of MyD88 and IL-18R are the most effective in inhibiting homodimerization with either the isolated TIR or full-length MyD88. Moreover, we demonstrated that a cell permeable analog of MyD88 epta-peptide inhibits homodimerization of MyD88 TIR domains in an in vitro cell system and significantly reduces IL-1 signaling, as assayed by activation of the downstream transcription factor NF-B. Our results indicate that the BB-loop in TIR domain of MyD88 is a good target for specific inhibition of MyD88-mediated signaling in vivo.Myeloid differentiation factor 88 (MyD88) 1 is a crucial adaptor protein that functions to recruit signaling proteins to receptors of the Toll-like or interleukin-1 receptor (TLR/IL-1R) superfamily (1, 2). Activation of signaling pathways downstream of this class of receptors is fundamental for several aspects of host defense.The MyD88 protein has a modular structure composed of a death domain (DD) at the N terminus and a Toll/IL-1 receptor (TIR) domain at the C terminus separated by a short linker region, referred to as intermediary domain (3). Upon ligand stimulation, MyD88 is recruited to the membrane by interaction of its TIR domain with the analogous domain in the IL-1R or TLR receptors (4). It has been shown that MyD88 forms homodimers (5) and promotes the recruitment to the plasma membrane and the activation of two IL-1 receptor-associated kinases: IRAK-4 and IRAK-1. A homophilic interaction between MyD88 DD and the homologous DD found at the N terminus of the kinases is required for such event (6). A recent model proposes that MyD88 binds to IRAK-4 and promotes phoshorylation of critical IRAK-1 residues by IRAK-4 (7). These events stimulate IRAK-1 autophosphorylation and its interaction with TRAF6 (tumor necrosis factor (TNF) receptorassociated factor 6), leading to activation of both the inhibitory B kinase (IKK) and the mitogen-activated protein kinases (MAPK) JNK and p38. These kinases are pivotal in the ultimate activation of several transcription factors, including NF-B and activator protein 1 (AP-1), which elicit the production of essential effector molecules for immune and inflammatory responses (8). The generation of MyD88-deficient mice (9) has shown that this protein is required for the proliferative response of T-cell...

MyD88 is an adaptor protein, which plays an essential role in the intracellular signaling elicited by IL-1R and several TLRs. Central to its function is the ability of its Toll/IL-1R translation initiation region (TIR) domain to heterodimerize with the receptor and to homodimerize with another MyD88 molecule to favor the recruitment of downstream signaling molecules such as the serine/threonine kinases IL-1R-associated kinase 1 (IRAK1) and IRAK4. Herein, we have synthesized and tested the activity of a synthetic peptido-mimetic compound (ST2825) modeled after the structure of a heptapeptide in the BB-loop of the MyD88-TIR domain, which interferes with MyD88 signaling. ST2825 inhibited MyD88 dimerization in coimmunoprecipitation experiments. This effect was specific for homodimerization of the TIR domains and did not affect homodimerization of the death domains. Moreover, ST2825 interfered with recruitment of IRAK1 and IRAK4 by MyD88, causing inhibition of IL-1beta-mediated activation of NF-kappaB transcriptional activity. After oral administration, ST2825 dose-dependently inhibited IL-1beta-induced production of IL-6 in treated mice. Finally, we observed that ST2825 suppressed B cell proliferation and differentiation into plasma cells in response to CpG-induced activation of TLR9, a receptor that requires MyD88 for intracellular signaling. Our results indicate that ST2825 blocks IL-1R/TLR signaling by interfering with MyD88 homodimerization and suggest that it may have therapeutic potential in treatment of chronic inflammatory diseases.

Deregulation of the phosphatidyl inositol trisphosphate kinase/AKT/mammalian target of rapamycin (mTOR) and RAS/mitogen-activated protein kinase (MAPK)/MNK pathways frequently occurs in human prostate carcinomas (PCas) and leads to aberrant modulation of messenger RNA (mRNA) translation. We have investigated the relative contribution of these pathways to translational regulation and proliferation of PCa cells. MNK-dependent phosphorylation of eIF4E is elevated in DU145 cells, which have low basal levels of AKT/mTOR activity due to the expression of the tumor suppressor PTEN. In contrast, eIF4E phosphorylation is low in PC3 and LNCaP cells with mutated PTEN and constitutively active AKT/mTOR pathway, but it can be strongly induced through inhibition of mTOR activity by rapamycin or serum depletion. Remarkably, we found that inhibition of MNKs strongly reduced the polysomal recruitment of terminal oligopyrimidine messenger RNAs (TOP mRNAs), which are known targets of mTOR-dependent translational control. Pull-down assays of the eIF4F complex indicated that translation initiation was differently affected by inhibition of MNKs and mTOR. In addition, concomitant treatment with MNK inhibitor and rapamycin exerted additive effects on polysomal recruitment of TOP mRNAs and protein synthesis. The MNK inhibitor was more effective than rapamycin in blocking proliferation of PTEN-expressing cells, whereas combination of the two inhibitors suppressed cell cycle progression in both cell lines. Microarray analysis showed that MNK affected translation of mRNAs involved in cell cycle progression. Thus, our results indicate that a balance between the activity of the AKT/mTOR and the MAPK/MNK pathway in PCa cells maintains a defined translational level of specific mRNAs required for ribosome biogenesis, cell proliferation and stress response and might confer to these cells the ability to overcome negative insults.

The members of Toll-like receptor/Interleukin (IL)-1 receptor (TLR/IL-1R) superfamily play a fundamental role in the immune response. These receptors detect microbial components and trigger complex signalling pathways that result in increased expression of multiple inflammatory genes. On the other hand, an aberrant activation of TLR/IL-1R signalling can promote the onset of inflammatory and autoimmune diseases, raising the interest in the development of therapeutic strategies for the control of their function. In this review, we illustrate the structural and functional features of TLR/IL-1R proteins and discuss some recent advances in the approaches undertaken to develop anti-inflammatory therapeutic drugs. In particular, we will focus on inhibitors, such as decoy peptides and synthetic mimetics, that interfere with protein-protein interactions between signalling molecules of the TLR/IL-1R superfamily. Given their central role in innate and adaptive immune responses, it is foreseen that pharmaceutical modulation of TLR/IL-1R signalling pathways by these drugs might yield clinical benefits in the treatment of inflammatory and autoimmune diseases.