Maria Loiarro

Peptide-mediated Interference of TIR Domain Dimerization in MyD88 Inhibits Interleukin-1-dependent Activation of NF-κB

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-κB. 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.

Pivotal Advance: Inhibition of MyD88 dimerization and recruitment of IRAK1 and IRAK4 by a novel peptidomimetic compound.

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‐1β‐mediated activation of NF‐κB transcriptional activity. After oral administration, ST2825 dose‐dependently inhibited IL‐1β‐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.

Phosphorylation of eIF4E by MNKs supports protein synthesis, cell cycle progression and proliferation in prostate cancer cells

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.

Pharmacological inhibition of TLR9 activation blocks autoantibody production in human B cells from SLE patients

OBJECTIVES Toll-like receptor 9 (TLR9), which recognizes hypomethylated DNA [cytosine-phosphate-guanine (CpG)], plays a role in the maintenance of serological memory and has been recently implicated in the pathogenesis of SLE. We previously reported that in vitro TLR9 triggers memory B-cell differentiation into antibody-producing cells, and that the MyD88-inhibitor ST2825 blocks TLR9-induced plasma cell (PC) generation. Here, we investigated whether memory B cells produce autoantibodies in SLE patients with active disease or in clinical remission, and whether ST2825 could inhibit PC generation in SLE patients. METHODS Peripheral blood mononuclear cells from 10 SLE patients in clinical remission and 2 with active SLE were cultured in the presence of CpG with or without ST2825. Phenotypical analysis of CpG-stimulated cells was performed by flow cytometry. Supernatants were collected to measure antibody production by ELISA and to detect autoantibodies by IF. RESULTS CpG-induced TLR9 stimulation caused autoantibody secretion in patients with active disease and in the majority of patients in clinical remission. Inhibition of MyD88 completely blocked the de novo generation of PCs and the secretion of autoantibodies. CONCLUSIONS Autoreactive B cells persist in SLE patients during disease remission in the circulating B-cell memory pool. TLR9-dependent activation of memory B cells by pathogens could be one of the mechanisms triggering relapses in SLE. Compounds targeting the TLR/MyD88 pathway may be used as novel therapeutic tools to treat acute disease and to prevent relapses in SLE patients.

Identification of critical residues of the MyD88 death domain involved in the recruitment of downstream kinases.

MyD88 couples the activation of the Toll-like receptors and interleukin-1 receptor superfamily with intracellular signaling pathways. Upon ligand binding, activated receptors recruit MyD88 via its Toll-interleukin-1 receptor domain. MyD88 then allows the recruitment of the interleukin-1 receptor-associated kinases (IRAKs). We performed a site-directed mutagenesis of MyD88 residues, conserved in death domains of the homologous FADD and Pelle proteins, and analyzed the effect of the mutations on MyD88 signaling. Our studies revealed that mutation of residues 52 (MyD88E52A) and 58 (MyD88Y58A) impaired recruitment of both IRAK1 and IRAK4, whereas mutation of residue 95 (MyD88K95A) only affected IRAK4 recruitment. Since all MyD88 mutants were defective in signaling, recruitment of both IRAKs appeared necessary for activation of the pathway. Moreover, overexpression of a green fluorescent protein (GFP)-tagged mini-MyD88 protein (GFP-MyD88-(27–72)), comprising the Glu52 and Tyr58 residues, interfered with recruitment of both IRAK1 and IRAK4 by MyD88 and suppressed NF-κB activation by the interleukin-1 receptor but not by the MyD88-independent TLR3. GFP-MyD88-(27–72) exerted its effect by titrating IRAK1 and suppressing IRAK1-dependent NF-κB activation. These experiments identify novel residues of MyD88 that are crucially involved in the recruitment of IRAK1 and IRAK4 and in downstream propagation of MyD88 signaling.

17-β-estradiol elicits genomic and non-genomic responses in mouse male germ cells

Estrogens have been postulated to exert a detrimental effect on spermatogenesis in vivo. Since mouse male germ cells express estrogen receptors, we have investigated whether molecular pathways are activated by estrogen stimulation of these cells. Our results demonstrate that estrogen receptor β is expressed in mitotic and meiotic male germ cells as well as in the spermatogonia derived GC‐1 cell line. By using this cell line, we show that 17‐β‐estradiol triggers activation of a transcriptional response that requires a functional estrogen receptor. Moreover, GC‐1 cells respond to estrogens by transiently activating a signal transduction pathway that impinges on the mitogen‐activated protein kinases (MAPK) ERK1 and ‐2. A similar dose‐dependent transient activation of ERKs was also observed in primary mouse spermatocytes in culture. Activation by the estrogen was specific because other steroids such as progesterone and dihydrotestosterone were ineffective and because it could be blocked by the selective inhibitor of the ERK pathway and by competitive inhibitors of the estrogen receptor. Finally, we observed that 17‐β‐estradiol does not affect spontaneous or induced apoptosis in cultured mouse spermatocytes, indicating that the apoptotic effects observed in vivo require additional testicular components.

Mutational Analysis Identifies Residues Crucial for Homodimerization of Myeloid Differentiation Factor 88 (MyD88) and for Its Function in Immune Cells

Background: MyD88 is an adaptor protein that plays a crucial role in the immune response. Results: We identified residues within the TIR domain of MyD88 required for protein self-association. Conclusion: Interference with the surface of homodimerization identified by these residues inhibits MyD88 function. Significance: The inhibition of MyD88 activity could be a good therapeutic strategy for inflammatory and autoimmune diseases. Myeloid differentiation factor 88 (MyD88) is an adaptor protein that transduces intracellular signaling pathways evoked by the Toll-like receptors (TLRs) and interleukin-1 receptors (IL-1Rs). MyD88 is composed of an N-terminal death domain (DD) and a C-terminal Toll/IL-1 receptor (TIR) domain, separated by a short region. Upon ligand binding, TLR/IL-1Rs hetero- or homodimerize and recruit MyD88 through their respective TIR domains. Then, MyD88 oligomerizes via its DD and TIR domain and interacts with the interleukin-1 receptor-associated kinases (IRAKs) to form the Myddosome complex. We performed site-directed mutagenesis of conserved residues that are located in exposed regions of the MyD88-TIR domain and analyzed the effect of the mutations on MyD88 signaling. Our studies revealed that mutation of Glu183, Ser244, and Arg288 impaired homodimerization of the MyD88-TIR domain, recruitment of IRAKs, and activation of NF-κB. Moreover, overexpression of two green fluorescent protein (GFP)-tagged MyD88 mini-proteins (GFP-MyD88151–189 and GFP-MyD88168–189), comprising the Glu183 residue, recapitulated these effects. Importantly, expression of these dominant negative MyD88 mini-proteins competed with the function of endogenous MyD88 and interfered with TLR2/4-mediated responses in a human monocytic cell line (THP-1) and in human primary monocyte-derived dendritic cells. Thus, our studies identify novel residues of the TIR domain that are crucially involved in MyD88 homodimerization and TLR signaling in immune cells.

Targeting the Toll-like Receptor/Interleukin 1 Receptor Pathway in Human Diseases: Rational Design of MyD88 Inhibitors

Toll-like receptor (TLR)/interleukin (IL) 1 receptor (IL-1R) play a fundamental role in the immune response. These receptors are distributed in various cellular compartments and recognize different components of pathogens. All TLR/IL-1Rs, with the exception of TLR3, interact with MyD88, an intracellular adapter protein that triggers a signaling cascade that culminates in the expression of inflammatory genes. Because aberrant activation of TLR/IL-1Rs can promote the onset of inflammatory or autoimmune diseases and malignancies, this pathway has attracted considerable interest as a potential therapeutic target. Given the central role of MyD88 in TLR/IL-1R signaling, we set out different strategies to develop drugs that can block its function. Structural and functional analysis of the MyD88 domains allowed us to identify crucial residues required for MyD88 homodimerization. Moreover, we developed small cell-permeable peptides and peptidomimetic agents that inhibit MyD88 homodimerization and function. Our results pave the way for the development of new therapeutic drugs for the inhibition of MyD88-dependent signaling.

The p38 mitogen-activated protein kinase cascade modulates T helper type 17 differentiation and functionality in multiple sclerosis

The p38 mitogen‐activated protein kinase cascade is required for the induction of a T helper type 17 (Th17) ‐mediated autoimmune response, which underlies the development and progression of several autoimmune diseases, such as experimental autoimmune encephalomyelitis, the animal model of multiple sclerosis (MS). However, the contribution of p38 phosphorylation to human Th cell differentiation has not been clarified. Here we demonstrate that the p38 signalling pathway is implicated in the generation of Th17 lymphocytes from human CD4+ CD27+ CD45RA+ naive T cells, both in healthy donors and in patients affected by the relapsing–remitting form of MS. Our data also indicate that p38 activation is essential for interleukin‐17 release from central memory lymphocytes and committed Th17 cell clones. Furthermore, CD4+ T cells isolated from individuals with relapsing–remitting MS display an altered responsiveness of the p38 cascade, resulting in increased p38 phosphorylation upon stimulation. These findings suggest that the p38 signalling pathway, by modulating the Th17 differentiation and response, is involved in the pathogenesis of MS, and open new perspectives for the use of p38 inhibitors in the treatment of Th17‐mediated autoimmune diseases.

Anti-proliferative effect of a triazole derivative (ST1959) on LNCaP human prostate cancer cells through down-regulation of cyclin and androgen receptor expression.

Previous studies demonstrated that ST1959, a triazole derivative endowed with immunomodulatory activities, also exerts inhibitory effects on proliferation and survival of a panel of tumor cells. In this study, we sought to ascertain the effects of ST1959 on the growth of androgen‐dependent and androgen‐independent prostate cancer (PCa) cells.