Fas-associated protein with death domain (FADD) is a pivotal component of death receptormediated extrinsic apoptosis and necroptosis. Here we show that FADD is regulated by makorin Ring Finger Protein 1 (mKRn1) E3 ligase-mediated ubiquitination and proteasomal degradation. mKRn1 knockdown results in FADD protein stabilization and formation of the rapid deathinducing signalling complex, which causes hypersensitivity to extrinsic apoptosis by facilitating caspase-8 and caspase-3 cleavage in response to death signals. We also show that mKRn1 and FADD are involved in the regulation of necrosome formation and necroptosis upon caspase inhibition. Downregulation of mKRn1 results in severe defects of tumour growth upon tumour necrosis factor-related apoptosis-inducing ligand treatment in a xenograft model using mDAmB-231 breast cancer cells. suppression of tumour growth by mKRn1 depletion is relieved by simultaneous FADD knockdown. our data reveal a novel mechanism by which fas-associated protein with death domain is regulated via an ubiquitination-induced degradation pathway. A poptosis, or programmed cell death, can be initiated by intrinsic or extrinsic pathways 1,2 . Intrinsic pathways are triggered by mitochondrial permeabilization, which results in the release of cytochrome c and the formation of an apoptosome complex, leading to caspase-9 activation 3 . Extrinsic pathways can be induced by activation of death receptors (DRs), such as TNFR1, tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors and Fas/CD95 by their corresponding ligands; receptor activation triggers the formation of the death-inducing signalling complex (DISC) or TNFR complex II, which comprises caspase-8 and fas-associated protein with death domain (FADD). These complexes ultimately activate caspase-8, thereby causing extrinsic apoptosis 3,4 . In addition to inducing extrinsic apoptosis, caspase-8 and FADD are known to suppress necroptosis under various conditions [5][6][7][8][9][10][11] .DR downstream pathways are regulated in various ways. For example, cFLIP (cellular FLICE inhibitory protein) competes with caspase-8 for binding to FADD, preventing DISC formation 12 . The levels and activities of cFLIP are controlled by numerous factors including NF-κB, Akt, JNK, Srk and ITCH [13][14][15][16][17] . CARP-1 and -2, which are E3 ubiquitin ligases for caspase-8, are also known to suppress TRAIL activation 18 . Post-translational modifications such as O-glycosylation, S-nitrosylation and S-palmitoylation of DRs have also been identified to regulate death signalling 19 . However, phosphorylation of FADD, the only post-translational modification that FADD is known to undergo, is not involved in the induction of apoptosis. Rather, this modification seems to be essential for the pro-survival roles of nuclear-localized phospho-FADD [20][21][22][23] . Other post-translational modifications affecting the apoptotic and necroptotic activities of FADD have yet to be identified.Here, we show that Makorin Ring Finger Protein 1 (MKRN1), an...

Adipocytes are differentiated by various transcriptional cascades integrated on the master regulator, Pparγ. To discover new genes involved in adipocyte differentiation, preadipocytes were treated with three newly identified pro-adipogenic small molecules and GW7845 (a Pparγ agonist) for 24 hours and transcriptional profiling was analyzed. Four genes, Peroxisome proliferator-activated receptor γ (Pparγ), human complement factor D homolog (Cfd), Chemokine (C-C motif) ligand 9 (Ccl9), and GIPC PDZ Domain Containing Family Member 2 (Gipc2) were induced by at least two different small molecules but not by GW7845. Cfd and Ccl9 expressions were specific to adipocytes and they were altered in obese mice. Small hairpin RNA (shRNA) mediated knockdown of Cfd in preadipocytes inhibited lipid accumulation and expression of adipocyte markers during adipocyte differentiation. Overexpression of Cfd promoted adipocyte differentiation, increased C3a production, and led to induction of C3a receptor (C3aR) target gene expression. Similarly, treatments with C3a or C3aR agonist (C4494) also promoted adipogenesis. C3aR knockdown suppressed adipogenesis and impaired the pro-adipogenic effects of Cfd, further suggesting the necessity for C3aR signaling in Cfd-mediated pro-adipogenic axis. Together, these data show the action of Cfd in adipogenesis and underscore the application of small molecules to identify genes in adipocytes.

The clinical manifestations of West Nile virus (WNV), a member of the Flavivirus family, include febrile illness, sporadic encephalitis, and paralysis. The capsid (Cp) of WNV is thought to participate in these processes by inducing apoptosis through mitochondrial dysfunction and activation of caspase-9 and caspase-3. To further identify the molecular mechanism of the WNV capsid protein (WNVCp), yeast two-hybrid assays were employed using WNV-Cp as bait. Jab1, the fifth subunit of the COP9 signalosome, was subsequently identified as a molecule that interacts with WNVCp. Immunoprecipitation and glutathione S-transferase pulldown assays confirmed that direct interaction could occur between WNVCp and Jab1. Immunofluorescence microscopy demonstrated that the overexpressed WNVCp, which localized to the nucleolus, was translocated to the cytoplasm upon its co-expression with Jab1. When treated with leptomycin B, Jab1-facilitated nuclear exclusion of WNVCp was prevented, which indicated that the CRM1 complex is required for Jab1-facilitated nuclear export of WNVCp. Moreover, Jab1 promoted the degradation of WNVCp in a proteasome-dependent way. Consistent with this, WNVCpmediated cell cycle arrest at the G 2 phase in H1299 was prevented by exogenous Jab1. Finally, an analysis of WNVCp deletion mutants indicated that the first 15 amino acids were required for interaction with Jab1. Furthermore, the double-point mutant of the WNVCp, P5A/P8A, was incapable of binding to Jab1. These results indicate that Jab1 has a potential protective effect against pathogenic WNVCp and might provide a novel target site for the treatment of disease caused by WNV.

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Gleditsia sinensis thorns (GST) have been used as a traditional medicine for carbuncles and skin diseases. The purpose of this study was to decide whether non-toxicological levels of water extract of GST (WEGST) are effective in inhibiting the progress of prostate cancer formation and to identify the target molecule involved in the WEGST-mediated inhibitory process of prostate cancer cell migration and in vivo tumor formation. Through the Boyden chamber migration assay, we found that non-toxic levels of WEGST could not attenuate the PC3 migration to the bottom area coated with serum but significantly inhibited PC3 cell migration to the collagen-coated bottom area. We also found that non-toxic levels of WEGST significantly attenuated collagen against adhesion. Interestingly, ectopic administration of WEGST could not affect the expression of α2β1 integrin, which is known as a receptor of collagen. However, when the PC3 cells adhered to a collagen-coated plate, the expression of α2 integrin but not that of β1 integrin was significantly inhibited by the administration of non-toxic levels of WEGST, leading to the inhibition of focal adhesion kinase (FAK) phosphorylation. Furthermore, oral administration of WEGST (25 mg/kg/day) significantly inhibited the size of a PC3 cell-xenografted tumor. Taken together, these results suggest a novel molecular mechanism for WEGST to inhibit prostate cancer progression at particular stages, such as collagen-mediated adhesion and migration, and it might provide further development for the therapeutic use of WEGST in the treatment of prostate cancer progression.