Role of cysteine residues of p65/NF-kappaB on the inhibition by the sesquiterpene lactone parthenolide and N-ethyl maleimide, and on its transactivating potential.

Sesquiterpene lactones (SLs) are potent anti-inflammatory substances. It was previously shown that the anti-inflammatory effect could be partly explained by the inhibition of the transcription factor NF-kappaB. Whether they inhibit the DNA binding of NF-kappaB, the activation of the IkappaB-kinase, or both is still a matter of debate. The data supporting these hypotheses were obtained using different cell systems. In this contribution we analyzed the mechanism of the sesquiterpene lactone-mediated inhibition using different cell systems, and showed that in all the cell lines analyzed, SLs inhibited both NF-kappaB binding and the IkappaB-kinase, but that the former played a more preponderant role in the inhibition. These results again confirm the importance of cysteine 38 in the inhibition and regulation of NF-kappaB's function. Moreover, we compared the selectivity of the SL parthenolide with that of N-ethyl maleimide (NEM). We showed that NEM directly alkylated p65 as well as p50 of NF-kappaB, whereas SLs possess a selectivity towards p65. Finally, we studied the transactivating properties of various p65 mutants, to analyze the effect of exchanged cysteine residues in the DNA binding domain of NF-kappaB/p65 on its function and demonstrated that the transactivating potential of the mutants did not correlate with their DNA binding strenght.

[1]  H. Pahl.  Activators and target genes of Rel/NF-κB transcription factors , 1999, Oncogene.

[2]  P. Tak,et al.  NF-κB: a key role in inflammatory diseases , 2001 .

[3]  Pei-ji Chen,et al.  An exceptionally well-preserved theropod dinosaur from the Yixian Formation of China , 1998, Nature.

[4]  M. Schmitz,et al.  I?B-independent control of NF-?B activity by modulatory phosphorylations , 2001 .

[5]  L. Hsu,et al.  Promoter clearance and escape in prokaryotes. , 2002, Biochimica et biophysica acta.

[6]  G. Ghosh,et al.  Crystal structure of p50/p65 heterodimer of transcription factor NF-κB bound to DNA , 1998, Nature.

[7]  G. Natoli,et al.  Anti-inflammatory cyclopentenone prostaglandins are direct inhibitors of IκB kinase , 2000, Nature.

[8]  H. Pahl,et al.  Cysteine 38 in p65/NF-κB Plays a Crucial Role in DNA Binding Inhibition by Sesquiterpene Lactones* , 2001, The Journal of Biological Chemistry.

[9]  Xianglin Shi,et al.  New insights into the role of nuclear factor-kappaB, a ubiquitous transcription factor in the initiation of diseases. , 1999, Clinical chemistry.

[10]  R. Bauer,et al.  Phytomedicines of Europe : chemistry and biological activity , 1998 .

[11]  F. Graham,et al.  Characteristics of a human cell line transformed by DNA from human adenovirus type 5. , 1977, The Journal of general virology.

[12]  L. O’Neill,et al.  Studies into the Effect of the Tyrosine Kinase Inhibitor Herbimycin A on NF-κB Activation in T Lymphocytes EVIDENCE FOR COVALENT MODIFICATION OF THE p50 SUBUNIT (*) , 1995, The Journal of Biological Chemistry.

[13]  E. Davioud‐Charvet,et al.  The conserved redox-sensitive cysteine residue of the DNA-binding region in the c-Rel protein is involved in the regulation of the phosphorylation of the protein. , 2000, The Biochemical journal.

[14]  H. Pahl,et al.  Inhibition of transcription factor NF-kappaB by sesquiterpene lactones: a proposed molecular mechanism of action. , 1999, Bioorganic & medicinal chemistry.

[15]  G. Willuhn.  ArnicaFlowers: Pharmacology, Toxicolgy, and Analysis of the Sesquiterpene Lactones—Their Main Active Substances , 1998 .

[16]  P. Klatt,et al.  Glutathionylation of the p50 subunit of NF-kappaB: a mechanism for redox-induced inhibition of DNA binding. , 2001, Biochemistry.

[17]  J. Yodoi,et al.  Thiordoxin regulates the DNA binding activity of NF-χB by reduction of a disulphid bond involving cysteine 62 , 1992 .

[18]  R. Gaynor,et al.  Therapeutic potential of inhibition of the NF-kappaB pathway in the treatment of inflammation and cancer. , 2001, The Journal of clinical investigation.

[19]  S. Heptinstall,et al.  Feverfew: A Review of Its History, Its Biological and Medicinal Properties, and the Status of Commercial Preparations of the Herb , 1998 .

[20]  C. Gélinas,et al.  The RxxRxRxxC motif conserved in all Rel/kappa B proteins is essential for the DNA-binding activity and redox regulation of the v-Rel oncoprotein , 1992, Molecular and cellular biology.

[21]  H. Pahl,et al.  The Anti-inflammatory Sesquiterpene Lactone Helenalin Inhibits the Transcription Factor NF-κB by Directly Targeting p65* , 1998, The Journal of Biological Chemistry.

[22]  D. Baltimore,et al.  Activation of DNA-binding activity in an apparently cytoplasmic precursor of the NF-κB transcription factor , 1988, Cell.

[23]  P. Baeuerle,et al.  The p65 subunit is responsible for the strong transcription activating potential of NF‐kappa B. , 1991, The EMBO journal.

[24]  T. Hofmann,et al.  The Antiinflammatory Sesquiterpene Lactone Parthenolide Inhibits NF-κB by Targeting the IκB Kinase Complex , 1999, The Journal of Immunology.

[25]  G. Bornkamm,et al.  Characterization of EBV‐genome negative “null” and “T” cell lines derived from children with acute lymphoblastic leukemia and leukemic transformed non‐Hodgkin lymphoma , 1977, International journal of cancer.

[26]  M. Karin,et al.  Phosphorylation meets ubiquitination: the control of NF-[kappa]B activity. , 2000, Annual review of immunology.

[27]  T. Schmidt,et al.  Helenanolide-type sesquiterpene lactones--III. Rates and stereochemistry in the reaction of helenalin and related helenanolides with sulfhydryl containing biomolecules. , 1997, Bioorganic & medicinal chemistry.

[28]  B. Hwang,et al.  Kaurane Diterpene, Kamebakaurin, Inhibits NF-κB by Directly Targeting the DNA-binding Activity of p50 and Blocks the Expression of Antiapoptotic NF-κB Target Genes* , 2002, The Journal of Biological Chemistry.

[29]  P. Ralph,et al.  Antibody-dependent killing of erythrocyte and tumor targets by macrophage-related cell lines: enhancement by PPD and LPS. , 1977, Journal of immunology.

[30]  H. Pahl,et al.  Helenalin, an Anti-Inflammatory Sesquiterpene Lactone from Arnica, Selectively Inhibits Transcription Factor NF-κB , 1997 .

[31]  S. Ruben,et al.  Isolation of a rel-related human cDNA that potentially encodes the 65-kD subunit of NF-kappa B. , 1991, Science.

[32]  M Elofsson,et al.  The anti-inflammatory natural product parthenolide from the medicinal herb Feverfew directly binds to and inhibits IkappaB kinase. , 2001, Chemistry & biology.

[33]  Urban Mb,et al.  The role of the p50 and p65 subunits of NF-kappa B in the recognition of cognate sequences. , 1991 .

[34]  Michael Karin,et al.  NF-κB at the crossroads of life and death , 2002, Nature Immunology.

[35]  A. Dvir,et al.  Promoter escape by RNA polymerase II. , 2002, Biochimica et biophysica acta.

[36]  A. Israël,et al.  The DNA binding subunit of NF-κB is identical to factor KBF1 and homologous to the rel oncogene product , 1990, Cell.

[37]  E. Pineda‐Molina,et al.  15-Deoxy-Delta 12,14-prostaglandin J2 inhibition of NF-kappaB-DNA binding through covalent modification of the p50 subunit. , 2001, The Journal of biological chemistry.

[38]  G. Towers,et al.  Formation of adducts of parthenin and related sesquiterpene lactones with cysteine and glutathione. , 1979, Chemico-biological interactions.

[39]  H. Wong,et al.  Sesquiterpene lactones are potent inhibitors of interleukin 8 gene expression in cultured human respiratory epithelium. , 2000, Cytokine.

[40]  H. E. Marshall,et al.  Inhibition of NF-kappa B by S-nitrosylation. , 2001, Biochemistry.

[41]  Y. Ben-Neriah,et al.  Rapid proteolysis of IκB-α is necessary for activation of transcription factor NF-κB , 1993, Nature.

[42]  C. Glass,et al.  15-Deoxy-Δ12,14-prostaglandin J2 inhibits multiple steps in the NF-κB signaling pathway , 2000 .

[43]  P. Harper,et al.  George Otto Gey. (1899-1970). The HeLa cell and a reappraisal of its origin. , 1971, Obstetrics and gynecology.

[44]  Michael Karin,et al.  The IκB kinase (IKK) and NF-κB: key elements of proinflammatory signalling , 2000 .