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Transcript
Inhibition of NF-kB by ZAS3, a zinc-finger protein that also binds to the kB motif Authors: Joung-Woo Hong, Carl E. Allen, and Lai-Chu Wu Presenters: Melissa Sherman & Troy Williams Overview of NF-kB • NF-kB pathway is used mainly during inflammation and development • ZAS and NF-kB are two distinct families of kBbinding proteins • NF-kB is bound to inhibitory molecule IkB in the cytoplasm • Rel family of NF-kB encodes transcription factors that regulate genes involved in: – Immune responses – Inflammation responses – Antiapoptotic responses Overview of NF-kB cont. • Rel family of NF-kB differ from non-Rel-kBs by: – Size – Immunogenicity – Sequence specificity to the kB motif • Rel family interacts with the kB motif to induce gene expression • Rel family includes p65.p50 and p50.p50 dimers NF-kB p65 p50 kB motif = GGGACTTTCC Basic NF-kB Pathway • TNF is released • TNF receptors on target cell bind cytokine • Trimerization of TNF occurs • TRADD, RIP, and TRAF2 bind to receptors • IKKK is activated • IKK is phosphorylated • IkB is phophorylated • IkB releases NF-kB – IkB is ubiquitylated – NF-kB transported to nucleus • NF-kB binds to promoters (kB motif) and activates transcription Overview of ZAS • ZAS proteins are large zinc-finger transcriptional proteins used in: – Growth – Signal transduction – Lymphoid development • ZAS contains: – Pair of zinc fingers – Acidic region – Serine/threonine-rich sequence • A zinc finger is part of a protein that can bind to DNA Overview of ZAS cont. • • • • There are three ZAS proteins: ZAS1, 2, and 3 ZAS3 is present in B lymphocytes ZAS fusion proteins can bind to kB motif ZAS3 inhibits NF-kB activation by: – Inhibition of nuclear translocation of p65 – Competition for kB gene regulatory elements – Repression of target gene transcription Inhibited NF-kB Pathway • TNF is released and binds to TNF receptors • Trimerization occurs • TRADD, RIP, TRAF2, and FADD bind to receptors – FADD is an adaptor known to transmit apoptotic and cell proliferation signals • ZAS3 binds to TRAF2 preventing IKK complex – Nuclear translocation of p65 prevented • ZAS3 may compete for kB gene regulatory elements or repress transcription The Experiment • Previous studies show that ZAS3 associates with TRAF2 to inhibit NF-kB activation in cytoplasm • Does ZAS 3 inhibit NF-kB activation in the nucleus? • Observed that ZAS binds specifically to kB-like sequences • Are ZAS proteins non-Rel-kBs? • Show that non-Rel-kB is absent in ZAS3-/- cells • Show that NF-kB is expressed in ZAS3-/- cells • Mechanism provides a checkpoint to control the reprogramming of gene expression EMSAs • Electrophoretic Mobility-Shift Assays (EMSAs) • B lymphocyte cell lines • 32P-kB probe yielded several sequence-specific DNAprotein complexes (C1, C2, and C3) EMSAs • Addition of ZAS3 antiserum diminished C1 • p65 and p50 antibodies altered DNA-protein complex patterns –C2 likely p50.p50 –C3 likely p65.p50 •Unlabeled kB oligonucleotides diminished all complexes •Sp1 oligonucleotides were noncompetitive EMSAs • ZAS3 knocked out by homologous recombination (ZAS-/-B1 and ZAS3-/-B2) • Prominent p65.p50 complex observed • C1 was not observed • Conclusion: ZAS most likely C1 EMSAs • LPS is a reagent used to stimulate NF-kB in B lymphocytes •ZAS3-/- cells: NF-kB DNA-binding was mostly found in nuclear extracts • LPS had minimal effect •ZAS3+/+ cells: NF-kB DNA-binding was found in both nuclear and cytoplasmic extracts •LPS reduced cytoplasmic binding and increased nuclear binding Immunoblot • Nuclear p65 levels are 5 times higher in ZAS-/- cells than ZAS+/+ cells • LPS increased nuclear p65 levels significantly in ZAS+/+ cells, but minimally in ZAS -/- cells • Conclusion: Supports results of higher NF-kB DNAbinding activity in the nucleus Reporter Gene Assays • kB-reporter activity was 30fold higher in ZAS3-/- than ZAS+/+ • LPS increased kB-reporter activity heavily in 38B9 cells and minimally in ZAS-/- cells • Due in part to the upregulation of nuclear p65 • Conclusion: ZAS3 has major impact on NF-kB-dependent gene expression and cell fate Immunoblot • Amount of TRAF1 and TRAF2 was higher in ZAS3-/- cells than 38B9 cells • Conclusion: Expression of TRAF1 and TRAF2 controlled by NF-kB Immunoblot • Amount of IkB-alpha in 38B9 cells was significantly higher than that of ZAS3-/- cells • Conclusion: – In ZAS3-/- cells, the absence of ZAS3 activates the assembly of the IKK complex – IKK phosphorylates IkB, IkB and NF-kB separate, and IkB is ubiquitylated – In ZAS+/+ cells, IkB is never degraded and shows a higher concentration of IkB Immunoblot • A DNA fragment (with all of the structural parts of ZAS3) was inserted into a FLAG-tag expression plasmid. • It yielded abundant fusion proteins. • Conclusion: ZAS3 was also found more in the nucleus then the cytoplasm. Reporter Gene Assays • Fig. 3B- Reporter gene assays initially showed that ZAS3 expression reduced the activity of kB- reporter genes in HEK 293 • Fig. 3C- Expression constructs showed that ZAS3 repressed NF-kB-mediated transactivation of the kB-reporter. • Fig. 3D- ZAS3 expression also inhibited the kB reporter in ZAS3 -/- cells. • Conclusion: They all provide a link between ZAS3 deficiency and NF-kB activation. Immunoblot • In 38B9 cells, p65 was mainly located in the cytoplasmic extracts and barely in the nuclear ones • In ZAS3, p65 was mainly found in the nuclear extracts. • Introduction of ZAS3 expression construct caused an increase of expression in the cytoplasm and less activity in the nucleus. • H1 (a nuclear protein) was a control for the experiment • Conclusion: p65 was regulated in ZAS3 -/- cells, and that ZAS3 activated nuclear export pathway of p65 kB-Reporter Gene Assays • kB-reporter was much higher in p65+/+ cells – Demonstrates importance of p65 in kB-mediated transactiviation • When p65 was absent, kB-reporter gene activity was still higher than control – kB-reporter must be driven by transacting factors in kB-DNA • Conclusion: ZAS3 represses kBmediated transcription independent of p65 kB-Reporter Gene Assays • Expression vectors were cotransfected with reporter plasmid pCMV110, upstream CAT gene • Expression of the CAT reporter was increased 50-fold by plasmid pSGVP16 – VP16 domain is a strong transcriptional activator • Expression of the CAT reporter was repressed by plasmid pSG-ZASC • Conclusion: ZAS3 inhibits transactivation of VP16 and so may function as a transcriptional repressor Conclusions • Previous studies have determined ZAS helps to maintain normal growth. Expression of ZAS restricts proliferation and mitosis of germ cells • Down-regulation of ZAS is associated with accelerated growth of cell lines and growth of multinucleated giant cells • ZAS3 required maturation (prevents apoptosis) • In ZAS-/- cells, developmental abnormalities resulted including tumorigenesis, polydactyly, and hydronephrosis • Changes of ZAS gene expression have been associated with leukemia patients – Suggest ZAS3 contains tumor suppression function • ZAS proteins have been implicated in signal transduction • ZAS3 associates with TRAF2 to repress TNF/NF-kB pathway Conclusions cont. • Localization of p65 and transactivation activity of NFkB are modulated by ZAS3 • Where NF-kB activates transcription, ZAS3 mostly represses transcription • ZAS3 is responsible partly for guarding NF-kBmediated transactivation by: – Inhibiting nuclear localization of p65 – Competing for kB gene regulatory elements – Serving as a transcriptional repressor Conclusions cont. • ZAS family of zinc-finger proteins are governors of NF-kB-mediated cell functions including: – – – – Apoptosis Proliferation Inflammation Immunity • Non-Rel-kB most likely related to ZAS3