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1.
Mol Pharm ; 16(4): 1516-1522, 2019 04 01.
Article in English | MEDLINE | ID: mdl-30860380

ABSTRACT

Myeloid differentiation primary response 88 (MyD88) is an intracellular adaptor protein central to the signaling of multiple receptors involved in inflammation. Since innate immune inflammation promotes autoimmunity, MyD88 is an attractive target in autoimmune disease. We previously developed c(MyD 4-4), a novel cyclic peptide competitive inhibitor of MyD88 dimerization that is metabolically stable. Parenteral administration of c(MyD 4-4) reduces disease severity in a mouse model of the human autoimmune disease multiple sclerosis. We now show that N-terminal myristoylation of c(MyD 4-4) enhances the competitive inhibition of MyD88 dimerization in living cells, leading to improved inhibition of the Toll-like receptor and IL-1 receptor signaling. Importantly, myristoylation converts c(MyD 4-4) to an orally bioavailable inhibitor of MyD88. Oral administration of c(MyD 4-4) significantly lowered the inflammatory cytokines secreted by peripheral autoimmune T cells in mice immunized with myelin antigens and ameliorated disease severity in the mouse model of multiple sclerosis. Taken together, we show the conversion of a protein active region to a metabolically stable, selective cyclic peptide that is orally bioavailable.


Subject(s)
Myeloid Differentiation Factor 88/antagonists & inhibitors , Myristic Acid/metabolism , Peptides, Cyclic/administration & dosage , Peptides, Cyclic/chemistry , Toll-Like Receptors/metabolism , Administration, Oral , Animals , Biological Availability , Female , HEK293 Cells , Humans , Mice , Mice, Inbred C57BL , Myeloid Differentiation Factor 88/metabolism , Protein Processing, Post-Translational
2.
Sci Rep ; 8(1): 9476, 2018 06 21.
Article in English | MEDLINE | ID: mdl-29930295

ABSTRACT

MyD88 is a cytoplasmic adaptor protein that plays a central role in signaling downstream of the TLRs and the IL1R superfamily. We previously demonstrated that MyD88 plays a critical role in EAE, the murine model of multiple sclerosis, and showed that the MyD88 BB-loop decoy peptide RDVLPGT ameliorates EAE. We now designed and screened a library of backbone cyclized peptides based on the linear BB loop peptide, to identify a metabolically stable inhibitor of MyD88 that retains the binding properties of the linear peptide. We identified a novel cyclic peptide protein mimetic that inhibits inflammatory responses to TLR ligands, and NFκB activation in response to IL-1 activation. The inhibitor, c(MyD 4-4), is metabolically stable in comparison to the linear peptide, blocks MyD88 in a specific manner, and inhibits MyD88 function by preventing MyD88 dimerization. Finally, treatment of mice with c(MyD 4-4) reduced the severity of clinical disease in the murine EAE model of multiple sclerosis. Thus, modulation of MyD88-dependent signaling using c(MyD 4-4) is a potential therapeutic strategy to lower innate immune inflammation in autoimmune CNS disease.


Subject(s)
Anti-Inflammatory Agents/pharmacology , Encephalomyelitis, Autoimmune, Experimental/drug therapy , Myeloid Differentiation Factor 88/antagonists & inhibitors , Peptides, Cyclic/pharmacology , Animals , Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/therapeutic use , Binding Sites , Female , HEK293 Cells , HeLa Cells , Humans , Mice , Mice, Inbred C57BL , Myeloid Differentiation Factor 88/chemistry , Myeloid Differentiation Factor 88/metabolism , NF-kappa B/metabolism , Peptides, Cyclic/chemistry , Peptides, Cyclic/therapeutic use , Protein Binding , RAW 264.7 Cells , Receptors, Interleukin-1/metabolism , Toll-Like Receptors/metabolism
3.
PLoS Pathog ; 13(7): e1006472, 2017 Jul.
Article in English | MEDLINE | ID: mdl-28671993

ABSTRACT

Enteropathogenic Escherichia coli (EPEC), a common cause of infant diarrhea, is associated with high risk of mortality in developing countries. The primary niche of infecting EPEC is the apical surface of intestinal epithelial cells. EPEC employs a type three secretion system (TTSS) to inject the host cells with dozens of effector proteins, which facilitate attachment to these cells and successful colonization. Here we show that EPEC elicit strong NF-κB activation in infected host cells. Furthermore, the data indicate that active, pore-forming TTSS per se is necessary and sufficient for this NF-κB activation, regardless of any specific effector or protein translocation. Importantly, upon infection with wild type EPEC this NF-κB activation is antagonized by anti-NF-κB effectors, including NleB, NleC and NleE. Accordingly, this NF-κB activation is evident only in cells infected with EPEC mutants deleted of nleB, nleC, and nleE. The TTSS-dependent NF-κB activation involves a unique pathway, which is independent of TLRs and Nod1/2 and converges with other pathways at the level of TAK1 activation. Taken together, our results imply that epithelial cells have the capacity to sense the EPEC TTSS and activate NF-κB in response. Notably, EPEC antagonizes this capacity by delivering anti-NF-κB effectors into the infected cells.


Subject(s)
Enteropathogenic Escherichia coli/metabolism , Epithelial Cells/microbiology , Escherichia coli Infections/metabolism , Escherichia coli Infections/microbiology , Escherichia coli Proteins/metabolism , NF-kappa B/metabolism , Type III Secretion Systems/metabolism , Enteropathogenic Escherichia coli/genetics , Epithelial Cells/metabolism , Escherichia coli Infections/genetics , Escherichia coli Proteins/genetics , Host-Pathogen Interactions , Humans , NF-kappa B/genetics , Signal Transduction , Type III Secretion Systems/genetics
4.
Front Immunol ; 8: 615, 2017.
Article in English | MEDLINE | ID: mdl-28611775

ABSTRACT

Myeloid differentiation factor 88 (MyD88) recruits signaling proteins to the intracellular domain of receptors belonging to the toll-like/interleukin-1 (IL-1) receptor superfamily. Mice lacking MyD88 are highly susceptible to infectious diseases, but tend to resist experimentally induced autoimmune diseases such as experimental autoimmune encephalomyelitis (EAE) and manifest diminished allograft rejection. We reasoned that inhibition of MyD88 should influence the cytokine profile of responding T cells by blocking costimulatory molecule expression by antigen-presenting cells (APCs) and by inhibiting T-cell responses to IL-18. We now report that inhibition of MyD88 in human APCs led to decreased IFNγ and IL-17 production and a shift to IL-4 production by responding T cells in a mixed lymphocyte reaction. Direct inhibition of Myd88 in mouse and human T cells also reduced their production of IFNγ in response to IL-12/IL-18 stimulation. Finally, systemic MyD88 antagonism significantly reduced the clinical manifestations of EAE in mice. Thus, MyD88 appears to be a key factor in determining T cell phenotype and represents a potential target for therapeutic intervention.

5.
Cell Rep ; 18(2): 419-431, 2017 01 10.
Article in English | MEDLINE | ID: mdl-28076786

ABSTRACT

Whereas type I interferons (IFNs-I) were proposed to be elevated in human periodontitis, their role in the disease remains elusive. Using a bacterial-induced model of murine periodontitis, we revealed a prolonged elevation in IFN-I expression. This was due to the downregulation of TAM signaling, a major negative regulator of IFN-I. Further examination revealed that the expression of certain TAM components was reduced as a result of prolonged degradation of MYD88 by the infection. As a result of such prolonged IFN-I production, innate immunological functions of the gingiva were disrupted, and CD4+ T cells were constitutively primed by dendritic cells, leading to elevated RANKL expression and, subsequently, alveolar bone loss (ABL). Blocking IFN-I signaling restored proper immunological function and prevented ABL. Importantly, a loss of negative regulation on IFN-I expression by TAM signaling was also evident in periodontitis patients. These findings thus suggest a role for IFN-I in the pathogenesis of periodontitis.


Subject(s)
Interferon Type I/biosynthesis , Myeloid Differentiation Factor 88/metabolism , Porphyromonas gingivalis/physiology , Proteolysis , Receptors, Cell Surface/metabolism , Signal Transduction , Alveolar Bone Loss/complications , Alveolar Bone Loss/immunology , Alveolar Bone Loss/pathology , Animals , Bacteroidaceae Infections/complications , Bacteroidaceae Infections/immunology , Bacteroidaceae Infections/microbiology , Bone Resorption/complications , Bone Resorption/immunology , Bone Resorption/pathology , Dendritic Cells/immunology , Gingiva/microbiology , Gingiva/pathology , Humans , Interferon Type I/metabolism , Leukocytes/pathology , Lymph Nodes/pathology , Mice , Mouth Mucosa/microbiology , Mouth Mucosa/pathology , Periodontitis/immunology , Periodontitis/microbiology , Periodontitis/pathology
6.
J Histochem Cytochem ; 58(11): 1015-23, 2010 Nov.
Article in English | MEDLINE | ID: mdl-20713982

ABSTRACT

Serum amyloid A (SAA) is an acute phase protein which is expressed primarily in the liver as a part of the systemic response to various injuries and inflammatory stimuli; its expression in ovarian tumors has not been described. Here, we investigated the expression of SAA in human benign and malignant ovarian epithelial tumors. Non-radioactive in situ hybridization applied on ovarian paraffin tissue sections revealed mostly negative SAA mRNA expression in normal surface epithelium. Expression was increased gradually as epithelial cells progressed through benign and borderline adenomas to primary and metastatic adenocarcinomas. Similar expression pattern of the SAA protein was observed by immunohistochemical staining. RT-PCR analysis confirmed the overexpression of the SAA1 and SAA4 genes in ovarian carcinomas compared with normal ovarian tissues. In addition, strong expression of SAA mRNA and protein was found in the ovarian carcinoma cell line OVCAR-3. Finally, patients with ovarian carcinoma had high SAA serum levels, which strongly correlated with high levels of CA-125 and C-reactive protein. Enhanced expression of SAA in ovarian carcinomas may play a role in ovarian tumorigenesis and may have therapeutic application.


Subject(s)
Carcinoma/genetics , Carcinoma/pathology , Gene Expression Regulation, Neoplastic , Ovarian Neoplasms/genetics , Ovarian Neoplasms/pathology , Serum Amyloid A Protein/genetics , Serum Amyloid A Protein/metabolism , Adult , Aged , Aged, 80 and over , C-Reactive Protein/metabolism , CA-125 Antigen/blood , Carcinoma/blood , Cell Line, Tumor , Female , Humans , Middle Aged , Neoplasm Metastasis , Ovarian Neoplasms/blood , Ovary/cytology , Ovary/metabolism , Ovary/pathology , Reverse Transcriptase Polymerase Chain Reaction , Young Adult
7.
Biochem Biophys Res Commun ; 368(2): 368-73, 2008 Apr 04.
Article in English | MEDLINE | ID: mdl-18237545

ABSTRACT

We have recently reported that the acute phase protein serum amyloid A (SAA), is locally and differentially expressed in neoplastic tissues of human colon. In the present study, we demonstrate that SAA enhances the plasminogen activation (PA)-activity of HT-29 colon cancer cell line. Cell-associated PA-activity was measured following the plasminogen-dependent ability of the cells to cleave the chromogenic substrate S-2251. The SAA-enhanced PA-activity was inhibited by anti-SAA antibodies. These antibodies also decreased the basal PA-activity of HT-29 cells and neutralized their cytokines (Interleukin-1beta+Interleukin-6)-enhanced PA-activity. Using specific chromogenic substrates and the fibrin clot-lysis assay, we found that SAA enhances also the PA-activity mediated by purified urokinase- and tissue-type plasminogen activators. Together, the data indicate that SAA enhances plasminogen activation and suggest its possible role in plasmin(ogen)-mediated colon cancer progression.


Subject(s)
Colonic Neoplasms/metabolism , Fibrinolysin/metabolism , Plasminogen Activators/metabolism , Plasminogen/metabolism , Serum Amyloid A Protein/metabolism , HT29 Cells , Humans
8.
J Histochem Cytochem ; 54(1): 63-73, 2006 Jan.
Article in English | MEDLINE | ID: mdl-16116035

ABSTRACT

Serum amyloid A (SAA) is an acute phase reactant, whose level in the blood is elevated in response to trauma, infection, inflammation, and neoplasia. Elevated levels of SAA in the serum of cancer patients were suggested to be of liver origin rather than a tumor cell product. The role of SAA in human malignancies has not been elucidated. We investigated the expression of SAA at various stages of human colon carcinoma progression. Nonradioactive in situ hybridization applied on paraffin tissue sections from 26 colon cancer patients revealed barely detected SAA mRNA expression in normal looking colonic epithelium. Expression was increased gradually as epithelial cells progressed through dysplasia to neoplasia. Deeply invading colon carcinoma cells showed the highest levels of SAA. Expression was also found in colon carcinoma metastases. Cells of lymphoid follicles of the intestinal wall, inflammatory cells, ganglion cells, and endothelial cells, also expressed SAA mRNA. Immunohistochemical staining revealed SAA protein expression that colocalized with SAA mRNA expression. RT-PCR analysis confirmed the expression of the SAA1 and SAA4 genes in colon carcinomas, expression that was barely detectable in normal colon tissues. These findings indicate local and differential expression of SAA in human colon cancer tissues and suggest its role in colonic tumorigenesis.


Subject(s)
Colon/metabolism , Colonic Neoplasms/metabolism , Intestinal Mucosa/metabolism , Precancerous Conditions/metabolism , Serum Amyloid A Protein/biosynthesis , Adenocarcinoma/metabolism , Adenocarcinoma/secondary , Adenomatous Polyps/metabolism , Aged , Aged, 80 and over , Colonic Neoplasms/pathology , Female , Humans , Immunohistochemistry , In Situ Hybridization , Liver Neoplasms/metabolism , Liver Neoplasms/secondary , Lymph Nodes/metabolism , Lymphatic Metastasis , Male , Middle Aged , Omentum/metabolism , Peritoneal Neoplasms/metabolism , Peritoneal Neoplasms/secondary , RNA, Messenger/biosynthesis , Reverse Transcriptase Polymerase Chain Reaction , Serum Amyloid A Protein/genetics
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