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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...

Variations in glycosylation levels or in the glycoprofile of a certain glycoprotein in tumor-related sera have been widely reported and can be used as a means of differentiation. However, quantitative mass analysis of glycoproteins is difficult because of their high structural complexity and low mass sensitivity of glycopeptides. Therefore, more powerful technologies are required for the discovery of these potential biomarkers. Tissue inhibitor of metalloproteinase 1 (TIMP1), a glycoprotein typically present at a low concentration in serum, is known to be aberrantly glycosylated in colorectal cancer cell lines as a result of the terminal addition of beta-1,6-N-acetylglucosamine (beta-1,6-GlcNAc) by N-acetylglucosaminyltransferase-V (GnT-V), which is reportedly up-regulated in invasive/metastatic cancer cells. In this report, a highly sensitive method is presented for the quantitative analysis of aberrant GlcNAcylated TIMP1 in the serum of colorectal cancer (CRC) patients. Glycoproteins having an N-linked glycan terminating with beta-1,6-GlcNAc were enriched by phytohemagglutinin-L(4) (L-PHA), a lectin that specifically recognizes the beta-1,6-GlcNAc moiety of N-linked glycan. The L-PHA-enriched glycoproteins were digested in solution with trypsin. With the use of a monoclonal anti-peptide TIMP1 antibody linked covalently to magnetic beads, a unique target peptide (antigen) of TIMP1 was immuno-enriched from the L-PHA-enriched tryptic digests and analyzed quantitatively by multiple reaction monitoring (MRM) mass analysis. The systematic coupling of L-PHA lectin enrichment followed by stable isotope standards and capture by anti-peptide antibodies (SISCAPA) with MRM mass analysis afforded quantitation of TIMP1 at attomolar (10(-18)) concentrations. An aberrantly GlcNAcylated substoichiometric TIMP1 isoform was quantified at approximately 0.8 ng/mL serum, using sample equivalent to only 1.7 microL of serum from a CRC patient. This approach provides a useful tool for the quantitation of a specific aberrant glycoform from human serum containing a variety of protein isoforms and may be helpful in studies of biological function as it pertains to protein glycan heterogeneity.