K63 ubiquitination in immune signaling.

Ubc13-catalyzed K63 ubiquitination is a major control point for immune signaling. Recent evidence has shown that the control of multiple immune functions, including chronic inflammation, pathogen responses, lymphocyte activation, and regulatory signaling, is altered by K63 ubiquitination. In this review, we detail the novel cellular sensors that are dependent on K63 ubiquitination for their function in the immune signaling network. Many pathogens, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), can target K63 ubiquitination to inhibit pathogen immune responses; we describe novel details of the pathways involved and summarize recent clinically relevant SARS-CoV-2-specific responses. We also discuss recent evidence that regulatory T cell (Treg) versus T helper (TH) 1 and TH17 cell subset regulation might involve K63 ubiquitination. Knowledge gaps that merit future investigation and clinically relevant pathways are also addressed.

Heterozygous calcyclin-binding protein/Siah1-interacting protein (CACYBP/SIP) gene pathogenic variant linked to a dominant family with paucity of interlobular bile duct.

Paucity of interlobular bile ducts (PILBD) is a heterogeneous disorder classified into two categories, syndromic and non-syndromic bile duct paucity. Syndromic PILBD is characterized by the presence of clinical manifestations of Alagille syndrome. Non-syndromic PILBD is caused by multiple diseases, such as metabolic and genetic disorders, infectious diseases, and inflammatory and immune disorders. We evaluated a family with a dominantly inherited PILBD, who presented with cholestasis at 1-2 months of age but spontaneously improved by 1 year of age. Next-generation sequencing analysis revealed a heterozygous CACYBP/SIP p.E177Q pathogenic variant. Calcyclin-binding protein and Siah1 interacting protein (CACYBP/SIP) form a ubiquitin ligase complex and induce proteasomal degradation of non-phosphorylated ß-catenin. Immunohistochemical analysis revealed a slight decrease in CACYBP and ß-catenin levels in the liver of patients in early infancy, which almost normalized by 13 months of age. The CACYBP/SIP p.E177Q pathogenic variant may form a more active or stable ubiquitin ligase complex that enhances the degradation of ß-catenin and delays the maturation of intrahepatic bile ducts. Our findings indicate that accurate regulation of the ß-catenin concentration is essential for the development of intrahepatic bile ducts and CACYBP/SIP pathogenic variant is a novel cause of PILDB.

SIP/CacyBP promotes autophagy by regulating levels of BRUCE/Apollon, which stimulates LC3-I degradation.

BRUCE/Apollon is a membrane-associated inhibitor of apoptosis protein that is essential for viability and has ubiquitin-conjugating activity. On initiation of apoptosis, the ubiquitin ligase Nrdp1/RNF41 promotes proteasomal degradation of BRUCE. Here we demonstrate that BRUCE together with the proteasome activator PA28γ causes proteasomal degradation of LC3-I and thus inhibits autophagy. LC3-I on the phagophore membrane is conjugated to phosphatidylethanolamine to form LC3-II, which is required for the formation of autophagosomes and selective recruitment of substrates. SIP/CacyBP is a ubiquitination-related protein that is highly expressed in neurons and various tumors. Under normal conditions, SIP inhibits the ubiquitination and degradation of BRUCE, probably by blocking the binding of Nrdp1 to BRUCE. On DNA damage by topoisomerase inhibitors, Nrdp1 causes monoubiquitination of SIP and thus promotes apoptosis. However, on starvation, SIP together with Rab8 enhances the translocation of BRUCE into the recycling endosome, formation of autophagosomes, and degradation of BRUCE by optineurin-mediated autophagy. Accordingly, deletion of SIP in cultured cells reduces the autophagic degradation of damaged mitochondria and cytosolic protein aggregates. Thus, by stimulating proteasomal degradation of LC3-I, BRUCE also inhibits autophagy. Conversely, SIP promotes autophagy by blocking BRUCE-dependent degradation of LC3-I and by enhancing autophagosome formation and autophagic destruction of BRUCE. These actions of BRUCE and SIP represent mechanisms that link the regulation of autophagy and apoptosis under different conditions.

Endoplasmic reticulum protein BI-1 regulates Ca²âº-mediated bioenergetics to promote autophagy.

Autophagy is a lysosomal degradation pathway that converts macromolecules into substrates for energy production during nutrient-scarce conditions such as those encountered in tumor microenvironments. Constitutive mitochondrial uptake of endoplasmic reticulum (ER) Ca²âº mediated by inositol triphosphate receptors (IP3Rs) maintains cellular bioenergetics, thus suppressing autophagy. We show that the ER membrane protein Bax inhibitor-1 (BI-1) promotes autophagy in an IP3R-dependent manner. By reducing steady-state levels of ER Ca²âº via IP3Rs, BI-1 influences mitochondrial bioenergetics, reducing oxygen consumption, impacting cellular ATP levels, and stimulating autophagy. Furthermore, BI-1-deficient mice show reduced basal autophagy, and experimentally reducing BI-1 expression impairs tumor xenograft growth in vivo. BI-1's ability to promote autophagy could be dissociated from its known function as a modulator of IRE1 signaling in the context of ER stress. The results reveal BI-1 as a novel autophagy regulator that bridges Ca²âº signaling between ER and mitochondria, reducing cellular oxygen consumption and contributing to cellular resilience in the face of metabolic stress.

Orphan Nuclear Receptor NR4A1 Binds a Novel Protein Interaction Site on Anti-apoptotic B Cell Lymphoma Gene 2 Family Proteins.

B cell lymphoma gene 2 (Bcl-2) family proteins are key regulators of programmed cell death and important targets for drug discovery. Pro-apoptotic and anti-apoptotic Bcl-2 family proteins reciprocally modulate their activities in large part through protein interactions involving a motif known as BH3 (Bcl-2 homology 3). Nur77 is an orphan member of the nuclear receptor family that lacks a BH3 domain but nevertheless binds certain anti-apoptotic Bcl-2 family proteins (Bcl-2, Bfl-1, and Bcl-B), modulating their effects on apoptosis and autophagy. We used a combination of NMR spectroscopy-based methods, mutagenesis, and functional studies to define the interaction site of a Nur77 peptide on anti-apoptotic Bcl-2 family proteins and reveal a novel interaction surface. Nur77 binds adjacent to the BH3 peptide-binding crevice, suggesting the possibility of cross-talk between these discrete binding sites. Mutagenesis of residues lining the identified interaction site on Bcl-B negated the interaction with Nur77 protein in cells and prevented Nur77-mediated modulation of apoptosis and autophagy. The findings establish a new protein interaction site with the potential to modulate the apoptosis and autophagy mechanisms governed by Bcl-2 family proteins.

ATG4B (Autophagin-1) phosphorylation modulates autophagy.

Autophagy is a catabolic cellular mechanism for entrapping cellular macromolecules and organelles in intracellular vesicles and degrading their contents by fusion with lysosomes. Important roles for autophagy have been elucidated for cell survival during nutrient insufficiency, eradication of intracellular pathogens, and counteracting aging through clearance of senescent proteins and mitochondria. Autophagic vesicles become decorated with LC3, a protein that mediates their fusion with lysosomes. LC3 is a substrate of the cysteine protease ATG4B (Autophagin-1), where cleavage generates a C-terminal glycine required for LC3 conjugation to lipids in autophagosomes. ATG4B both cleaves pro-LC3 and also hydrolyzes lipids from cleaved LC3. We show here that phosphorylation of ATG4B at Ser-383 and Ser-392 increases its hydrolyase activity as measured using LC3 as a substrate. Reconstituting atg4b(-/-) cells with phosphorylation-deficient ATG4B showed a role of ATG4B phosphorylation in LC3 delipidation and autophagic flux, thus demonstrating that the cellular activity of ATG4B is modulated by phosphorylation. Proteolytic conversion of pro-LC3 to LC3-I was not significantly impacted by ATG4B phosphorylation in cells. Phosphorylation-deficient ATG4B also showed reduced interactions with the lipid-conjugated LC3 but not unconjugated LC3. Taken together, these findings demonstrate a role for Ser-383 and Ser-392 phosphorylation of ATG4B in control of autophagy.

Vaccinia virus F1L protein promotes virulence by inhibiting inflammasome activation.

Host innate immune responses to DNA viruses involve members of the nucleotide-binding domain, leucine-rich repeat and pyrin domain containing protein (NLRP) family, which form "inflammasomes" that activate caspase-1, resulting in proteolytic activation of cytokines interleukin (IL)-1ß and IL-18. We hypothesized that DNA viruses would target inflammasomes to overcome host defense. A Vaccinia virus (VACV) B-cell CLL/lymphoma 2 (Bcl-2) homolog, F1L, was demonstrated to bind and inhibit the NLR family member NLRP1 in vitro. Moreover, infection of macrophages in culture with virus lacking F1L (ΔF1L) caused increased caspase-1 activation and IL-1ß secretion compared with wild-type virus. Virulence of ΔF1L virus was attenuated in vivo, causing altered febrile responses, increased proteolytic processing of caspase-1, and more rapid inflammation in lungs of infected mice without affecting cell death or virus replication. Furthermore, we found that a hexapeptide from F1L is necessary and sufficient for inhibiting the NLRP1 inflammasome in vitro, thus identifying a peptidyl motif required for binding and inhibiting NLRP1. The functional importance of this NLRP1-binding motif was further confirmed by studies of recombinant ΔF1L viruses reconstituted either with the wild-type F1L or a F1L mutant that fails to bind NLRP1. Cellular infection with wild-type F1L reconstituted virus-suppressed IL-1ß production, whereas mutant F1L did not. In contrast, both wild-type and mutant versions of F1L equally suppressed apoptosis. In vivo, the NLR nonbinding F1L mutant virus exhibited an attenuated phenotype similar to ΔF1L virus, thus confirming the importance of F1L interactions with NLRP1 for viral pathogenicity in mice. Altogether, these findings reveal a unique viral mechanism for evading host innate immune responses.

Novel phosphorylation and ubiquitination sites regulate reactive oxygen species-dependent degradation of anti-apoptotic c-FLIP protein.

The cytosolic protein c-FLIP (cellular Fas-associated death domain-like interleukin 1ß-converting enzyme inhibitory protein) is an inhibitor of death receptor-mediated apoptosis that is up-regulated in a variety of cancers, contributing to apoptosis resistance. Several compounds found to restore sensitivity of cancer cells to TRAIL, a TNF family death ligand with promising therapeutic potential, act by targeting c-FLIP ubiquitination and degradation by the proteasome. The generation of reactive oxygen species (ROS) has been implicated in c-FLIP protein degradation. However, the mechanism by which ROS post-transcriptionally regulate c-FLIP protein levels is not well understood. We show here that treatment of prostate cancer PPC-1 cells with the superoxide generators menadione, paraquat, or buthionine sulfoximine down-regulates c-FLIP long (c-FLIP(L)) protein levels, which is prevented by the proteasome inhibitor MG132. Furthermore, pretreatment of PPC-1 cells with a ROS scavenger prevented ubiquitination and loss of c-FLIP(L) protein induced by menadione or paraquat. We identified lysine 167 as a novel ubiquitination site of c-FLIP(L) important for ROS-dependent degradation. We also identified threonine 166 as a novel phosphorylation site and demonstrate that Thr-166 phosphorylation is required for ROS-induced Lys-167 ubiquitination. The mutation of either Thr-166 or Lys-167 was sufficient to stabilize c-FLIP protein levels in PPC-1, HEK293T, and HeLa cancer cells treated with menadione or paraquat. Accordingly, expression of c-FLIP T166A or K167R mutants protected cells from ROS-mediated sensitization to TRAIL-induced cell death. Our findings reveal novel ROS-dependent post-translational modifications of the c-FLIP protein that regulate its stability, thus impacting sensitivity of cancer cells to TRAIL.

A novel mouse model for investigating α-synuclein aggregates in oligodendrocytes: implications for the glial cytoplasmic inclusions in multiple system atrophy.

The aggregated alpha-synuclein (αsyn) in oligodendrocytes (OLGs) is one of the pathological hallmarks in multiple system atrophy (MSA). We have previously reported that αsyn accumulates not only in neurons but also in OLGs long after the administration of αsyn preformed fibrils (PFFs) in mice. However, detailed spatial and temporal analysis of oligodendroglial αsyn aggregates was technically difficult due to the background neuronal αsyn aggregates. The aim of this study is to create a novel mouse that easily enables sensitive and specific detection of αsyn aggregates in OLGs and the comparable analysis of the cellular tropism of αsyn aggregates in MSA brains. To this end, we generated transgenic (Tg) mice expressing human αsyn-green fluorescent protein (GFP) fusion proteins in OLGs under the control of the 2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNP) promoter (CNP-SNCAGFP Tg mice). Injection of αsyn PFFs in these mice induced distinct GFP-positive aggregates in the processes of OLGs as early as one month post-inoculation (mpi), and their number and size increased in a centripetal manner. Moreover, MSA-brain homogenates (BH) induced significantly more oligodendroglial αsyn aggregates than neuronal αsyn aggregates compared to DLB-BH in CNP-SNCAGFP Tg mice, suggestive of their potential tropism of αsyn seeds for OLGs. In conclusion, CNP-SNCAGFP Tg mice are useful for studying the development and tropism of αsyn aggregates in OLGs and could contribute to the development of therapeutics targeting αsyn aggregates in OLGs.

6-Deoxyjacareubin, a natural compound preventing hypoxia-induced cell death, ameliorates neurodegeneration in a mouse model of familial amyotrophic lateral sclerosis.

The central nervous system (CNS) uses a significant amount of oxygen for energy production. Decreased oxygen supply due to impaired blood supply critically damages the CNS. As chronic hypoxic conditions have diverse effects via the excessive production of reactive oxygen species, protection from hypoxic damage is important for cell survival. Recent studies have revealed that various markers of hypoxia are altered in age-related neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), indicating the involvement of hypoxia. However, therapeutic strategies targeting hypoxia-induced pathways in ALS have not been developed yet. We previously screened small-molecule compounds that inhibit hypoxia-induced cell death and identified 6-deoxyjacareubin. We hypothesized that the modulation of hypoxia signaling by 6-deoxyjacareubin might protect motor neurons in ALS. Here, we show that 6-deoxyjacareubin indeed ameliorates neurodegeneration in a mouse model of familial ALS. Administration of 6-deoxyjacareubin to this familial ALS model significantly attenuated disease progression and improved locomotor dysfunction. We also found that 6-deoxyjacareubin reduced motor neuron loss and glial activation. Our results indicate that 6-deoxyjacareubin might serve as a potential therapeutic tool for ALS. Moreover, these results suggest that modulation of hypoxia signaling pathways provides a promising strategy to develop therapies for other types of neurodegenerative diseases also characterized by hypoxia.

Mitochondrial dysfunction in a mouse model of prodromal Parkinson's disease: A metabolomic analysis.

For the development of disease-modifying therapies for Parkinson's disease (PD) the identification of biomarkers in the prodromal stage is urgently required. Because PD is considered a systemic disease even in the early stage, we performed a metabolomic analysis of the plasma from a mouse model of prodromal PD (p-PD). Increased levels of isobutyrylcarnitine in p-PD mice imply an abnormality in ß-oxidation in mitochondria, and increased levels of pyrimidine nucleoside can be associated with mitochondrial dysfunction. Consistent with these results, the immunoblot analysis showed a defect in mitochondrial complex I assembly in p-PD mice. These results suggest that systemic mitochondrial dysfunction may exist in p-PD mice and contribute to the pathogenesis of PD, potentially being useful as early biomarkers for PD.

Impact of Gba2 on neuronopathic Gaucher's disease and α-synuclein accumulation in medaka (Oryzias latipes).

Homozygous mutations in the lysosomal glucocerebrosidase gene, GBA1, cause Gaucher's disease (GD), while heterozygous mutations in GBA1 are a strong risk factor for Parkinson's disease (PD), whose pathological hallmark is intraneuronal α-synuclein (asyn) aggregates. We previously reported that gba1 knockout (KO) medaka exhibited glucosylceramide accumulation and neuronopathic GD phenotypes, including short lifespan, the dopaminergic and noradrenergic neuronal cell loss, microglial activation, and swimming abnormality, with asyn accumulation in the brains. A recent study reported that deletion of GBA2, non-lysosomal glucocerebrosidase, in a non-neuronopathic GD mouse model rescued its phenotypes. In the present study, we generated gba2 KO medaka and examined the effect of Gba2 deletion on the phenotypes of gba1 KO medaka. The Gba2 deletion in gba1 KO medaka resulted in the exacerbation of glucosylceramide accumulation and no improvement in neuronopathic GD pathological changes, asyn accumulation, or swimming abnormalities. Meanwhile, though gba2 KO medaka did not show any apparent phenotypes, biochemical analysis revealed asyn accumulation in the brains. gba2 KO medaka showed a trend towards an increase in sphingolipids in the brains, which is one of the possible causes of asyn accumulation. In conclusion, this study demonstrated that the deletion of Gba2 does not rescue the pathological changes or behavioral abnormalities of gba1 KO medaka, and GBA2 represents a novel factor affecting asyn accumulation in the brains.

Susceptibility to erastin-induced ferroptosis decreases during maturation in a human oligodendrocyte cell line.

Ferroptosis, a form of iron-dependent cell death caused by lipid peroxidation, has been implicated in neurological and other disorders. However, the mechanism of ferroptosis in oligodendrocytes is unclear. We tested the susceptibility of MO3.13 cells, an oligodendrocyte line, to ferroptosis after erastin treatment. Immature MO3.13 cells were more susceptible to erastin-induced ferroptosis than chemically differentiated mature MO3.13 cells. Increased expression of solute carrier family 7 member 11 (SLC7A11), which encodes a cystine/glutamate transporter, and greater glutathione concentrations were observed in mature compared with immature MO3.13 cells, linking glutathione to the resistance of mature MO3.13 cells to erastin-induced ferroptosis. These findings highlight the usefulness of immature MO3.13 cells in studies of ferroptosis and investigations into neuropathologies that involve oligodendrocytes.

BLOC1S2 interacts with the HIPPI protein and sensitizes NCH89 glioblastoma cells to apoptosis.

The HIPPI (HIP-1 protein interactor) protein is a multifunctional protein that is involved in the regulation of apoptosis. The interaction partners of HIPPI include HIP-1 (Huntingtin-interacting protein-1), Apoptin, Homer1c, Rybp/DEDAF, and BAR (bifunctional apoptosis regulator). In search for other binding partners of HIPPI, we performed a yeast two hybrid screen and identified BLOC1S2 (Biogenesis of lysosome-related organelles complex-1 subunit 2) as a novel HIPPI-interacting protein. In co-immunoprecipitation assays, BLOC1S2 specifically associates with HIPPI, but not with HIP-1. To study the expression of BLOC1S2 on the protein level, we generated a mouse monoclonal antibody specific for BLOC1S2 and a multiple tissue array comprising 70 normal and cancer tissue samples of diverse origin. BLOC1S2 protein is widely expressed in normal tissue as well as in malignant tumors with a tendency towards lower expression levels in certain subtypes of tumors. On the subcellular level, BLOC1S2 is expressed in an organellar-like pattern and co-localizes with mitochondria. Over-expression of BLOC1S2 in the presence or absence of HIPPI does not induce apoptosis. However, BLOC1S2 and HIPPI sensitize NCH89 glioblastoma cells to the pro-apoptotic actions of staurosporine and the death ligand TRAIL by enhancing caspase activation, cytochrome c release, and disruption of the mitochondrial membrane potential. Given its interaction with HIPPI and its pro-apoptotic activity, BLOC1S2 might play an important functional role in cancer and neurodegenerative diseases.

KCTD5, a putative substrate adaptor for cullin3 ubiquitin ligases.

Potassium channel tetramerization domain (KCTD) proteins contain a bric-a-brac, tramtrak and broad complex (BTB) domain that is most similar to the tetramerization domain (T1) of voltage-gated potassium channels. Some BTB-domain-containing proteins have been shown recently to participate as substrate-specific adaptors in multimeric cullin E3 ligase reactions by recruiting proteins for ubiquitination and subsequent degradation by the proteasome. Twenty-two KCTD proteins have been found in the human genome, but their functions are largely unknown. In this study, we have characterized KCTD5, a new KCTD protein found in the cytosol of cultured cell lines. The expression of KCTD5 was upregulated post-transcriptionally in peripheral blood lymphocytes stimulated through the T-cell receptor. KCTD5 interacted specifically with cullin3, bound ubiquitinated proteins, and formed oligomers through its BTB domain. Analysis of the interaction with cullin3 showed that, in addition to the BTB domain, some amino acids in the N-terminus of KCTD5 are required for binding to cullin3. These findings suggest that KCTD5 is a substrate-specific adaptor for cullin3-based E3 ligases.

HBXIP, cellular target of hepatitis B virus oncoprotein, is a regulator of centrosome dynamics and cytokinesis.

Hepatitis B virus accounts for more than 1 million cancer deaths annually, but the mechanism by which this virus promotes hepatocellular carcinoma remains unclear. The hepatitis B virus genome encodes an oncoprotein, HBx, which binds various cellular proteins including HBXIP. We show here that HBXIP is a regulator of centrosome duplication, required for bipolar spindle formation in HeLa human carcinoma cells and primary mouse embryonic fibroblast cells. We found that most cells deficient in HBXIP arrest in prometaphase with monopolar spindles whereas HBXIP overexpression causes tripolar or multipolar spindles due to excessive centrosome replication. Additionally, a defect in cytokinesis was seen in HBXIP-deficient HeLa cells, with most cells failing to complete division and succumbing eventually to apoptosis. Expression of viral HBx in HeLa cells mimicked the effects of HBXIP overexpression, causing excessive centrosome replication, resulting in tripolar and multipolar spindles and defective cytokinesis. Immunolocalization and fluorescent protein tagging experiments showed that HBXIP associates with microtubules of dividing cells and colocalizes with HBx on centrosomes. Thus, viral HBx and its cellular target HBXIP regulate centrosome dynamics and cytokinesis affecting genetic stability. In vivo experiments using antisense oligonucleotides targeting HBXIP in a mouse model of liver regeneration showed a requirement for HBXIP for growth and survival of replicating hepatocytes. Thus, HBXIP is a critical regulator of hepatocyte cell growth in vivo, making it a strong candidate for explaining the tumorigenic actions of viral HBx.

Oxidized analogs of Di(1H-indol-3-yl)methyl-4-substituted benzenes are NR4A1-dependent UPR inducers with potent and safe anti-cancer activity.

Di(1H-indol-3-yl)(4-trifluoromethylphenyl)methane (DIM-Ph-4-CF3) is an analog of orphan nuclear receptor 4A1 (NR4A1) ligand cytosporone B. We have synthesized several oxidation products of DIM-Ph-4-CF3, focusing on analogs with electron-withdrawing or donating groups at their phenyl ring 4-positions, and examined their anti-cancer activity and mechanism-of-action. Mesylates (DIM-Ph-4-X+ OMs-s) having CF3, CO2Me and Cl groups were more effective inhibitors of cancer cell viability than their precursors. 19F NMR spectroscopy and differential scanning calorimetry strongly indicated interactions of DIM-Ph-4-CF3+ OMs- with the NR4A1 ligand binding domain, and compound-induced apoptosis of prostate cancer cells was dependent on NR4A1. DIM-Ph-4-CF3+ OMs- showed robust inhibition of LNCaP prostate cancer xenografts with no apparent toxicity. In vitro and in vivo, DIM-Ph-4-CF3+ OMs- activated proapoptotic unfolded protein response (UPR) signaling in prostate cancer cells. Independently of DIM-Ph-4-CF3+ OMs-, the bulk of NR4A1 localized to the cytoplasm in various cancer cell lines, suggesting a cytoplasmic mechanism-of-action of DIM-Ph-4-CF3+ OMs- in UPR induction and cell death. In summary, the data suggest that oxidized analogs of DIM-Ph-4-CF3 possess potent and safe anti-cancer activity which is mediated through UPR signaling downstream of NR4A1 binding.

Cellular FLIP inhibits beta-catenin ubiquitylation and enhances Wnt signaling.

Cellular FLIP (cFLIP) is a close homologue of caspase 8 without caspase activity that inhibits Fas signaling. The cFLIP protein is often expressed in human tumors and is believed to suppress antitumor immune responses involving the Fas system. Here, we report that a long form of cFLIP (cFLIP-L) inhibits beta-catenin ubiquitylation and increases endogenous cytosolic beta-catenin, which results in translocation of beta-catenin into nuclei and induction of beta-catenin-dependent gene expression in cFLIP-L-expressing cells. When cells stably expressing cFLIP-L were stimulated with Wnt3a, enhanced Wnt signaling was observed compared with the control cells. Conversely, depletion of endogenous cFLIP results in reduced Wnt signaling. Furthermore, cFLIP-L increases secondary-body axis formation when coinjected with suboptimal doses of beta-catenin into early Xenopus embryos. Down-regulation of FADD by RNA-mediated interference abolishes the beta-catenin-dependent gene expression induced by cFLIP-L. These results indicate that cFLIP-L, in cooperation with FADD, enhances canonical Wnt signaling by inhibiting proteasomal degradation of beta-catenin, thus suggesting an additional mechanism involved with tumorgenesis, in addition to inhibiting Fas signaling.

Yeast and mammalian two-hybrid systems for studying protein-protein interactions.

An important step in the analysis of protein function is identification of the interaction partners of each protein. The two-hybrid system has been widely used to identify and explore protein-protein interactions. By using various two-hybrid systems, numerous protein interactions that regulate apoptosis signaling have been discovered that reveal unexpected functions of cancer-relevant proteins. Methods for performing two-hybrid experiments using either yeast or mammalian cells will be described in this chapter.

Melanoma antigen A12 regulates cell cycle via tumor suppressor p21 expression.

Melanoma-associated antigen family A (MAGE-A) is a family of cancer/testis antigens that are expressed in malignant tumors but not in normal tissues other than the testes. MAGE-A12 is a MAGE-A family gene whose tumorigenic function in cancer cells remains unclear. Searches of the Oncomine and NextBio databases revealed that malignant tumors show up-regulation of MAGE-A12 mRNA relative to corresponding normal tissue. In PPC1 primary prostatic carcinoma cells and in HCT116 colorectal cancer cells (wild type and p53-depleted), MAGE-A12 gene knockdown using siRNA or shRNA diminishes cancer cell proliferation as assessed by cellular ATP levels, cell counting, and clonogenic assays. FACS analyses of annexin V-PI staining and DNA content show that MAGE-A12 knockdown causes G2/M arrest and apoptosis. In tumor xenografts of HCT116 cells, conditional knockdown of MAGE-A12 suppresses tumor growth. The depletion of MAGE-A12 leads to the accumulation of tumor suppressor p21 in PPC1, HCT116, and p53-depleted HCT116 cells. Conversely, CDKN1A knockdown partially rescues the viability of PPC1 cells transfected with siRNA targeting MAGE-A12, while p21 overexpression leads to proliferation arrest in PPC-1 cells. Furthermore, exogenous MAGE-A12 expression promotes the ubiquitination of p21. Our findings reveal that MAGE-A12 plays crucial roles in p21 stability and tumor growth, suggesting that MAGE-A12 could provide a novel target for cancer treatment.