A 24-h treatment with the cytokine tumor necrosis factor-alpha (TNF-alpha) suppresses transcription of E-box-driven clock genes (D-site albumin promoter binding protein, Dbp; Tyrotroph embryonic factor, Tef ; Hepatic leukemia factor, Hlf; Period homolog to Drosophila 1/2/3, Per1, Per2, and Per3) by yet unknown molecular mechanisms. The attenuation of clock genes has been suggested as a putative cause for the development of sickness behavior syndrome in infectious and autoimmune diseases. Here, the authors studied the effect of TNF-alpha at early time points (<3 h) on intracellular signaling events and clock gene expression in fibroblasts. Interaction of TNF-alpha with TNFR1 (Tnfrsf1a , CD120a, p55), but not TNFR2 (Tnfrsf1b, CD120b , p75), leads to fast downregulation of gene expression of Dbp and upregulation of negative regulators of the molecular clock, Per1 and Per2, Cryptochrome-1 (Cry1), and Differentiated embryo chondrocytes-1 (Dec1). Since the decrease of Dbp is also observed in cells deficient for Per1/Per2, Cry1/Cry2 , or Dec1, these genes are unlikely to be responsible for inhibition of Dbp. The early effect of TNF-alpha on the clock gene Per1 is dependent on p38, mitogen-activated protein kinase (MAPK), and/or calcium signaling, whereas the effect on Dbp is independent of p38 MAPK, but also involves calcium signaling. Both genes remain unaffected by the NF-kappaB and AP-1 pathway. Taken collectively these data show p38 MAPK- and calcium-dependent TNFR1-mediated transient increase of the negative regulator Per1 and an independent decrease of Dbp.
Prion diseases are untreatable neurodegenerative disorders characterized by accumulation of PrP(Sc), an aggregated isoform of the cellular prion protein (PrP(C)). We generated a library of PrP variants with random mutations in the helix-3 domain and screened for dominant-negative mutants (DNMs) that would inhibit replication of prions (the Rocky Mountain Laboratory strain) in infected N2a cells. Two of the identified PrP mutants, Q167R and Q218K, were already known to counteract prion replication, thereby validating the effectiveness of this approach. In addition, novel DNMs were found efficiently to antagonize PrP(Sc) propagation in cells. In contrast to Q167R and Q218K, the newly identified DNMs S221P and Y217C resided on the cell surface at a substantially lower level, suggesting that robust cell surface display of DNM might not be a general prerequisite for efficient prion antagonism. The newly identified DNMs point to useful target-selective therapeutic tools for the treatment of prion diseases.
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