MOAP-1-mediated dissociation of p62/SQSTM1 bodies releases Keap1 and suppresses Nrf2 signaling.

Nrf2 signaling is vital for protecting cells against oxidative stress. However, its hyperactivation is frequently found in liver cancer through excessive build-up of p62/SQSTM1 bodies that sequester Keap1, an adaptor of the E3-ubiquitin ligase complex for Nrf2. Here, we report that the Bax-binding protein MOAP-1 regulates p62-Keap1-Nrf2 signaling through disruption of p62 bodies. Upon induction of cellular stresses that stimulate formation of p62 bodies, MOAP-1 is recruited to p62 bodies and reduces their levels independent of the autophagy pathway. MOAP-1 interacts with the PB1-ZZ domains of p62 and interferes with its self-oligomerization and liquid-liquid phase separation, thereby disassembling the p62 bodies. Loss of MOAP-1 can lead to marked upregulation of p62 bodies, enhanced sequestration of Keap1 by p62 and hyperactivation of Nrf2 antioxidant target genes. MOAP-1-deficient mice exhibit an elevated tumor burden with excessive levels of p62 bodies and Nrf2 signaling in a diethylnitrosamine (DEN)-induced hepatocarcinogenesis model. Together, our data define MOAP-1 as a negative regulator of Nrf2 signaling via dissociation of p62 bodies.

Lgr5+ pericentral hepatocytes are self-maintained in normal liver regeneration and susceptible to hepatocarcinogenesis.

Emerging evidence suggests that hepatocytes are primarily maintained by self-renewal during normal liver homeostasis, as well as in response to a wide variety of hepatic injuries. However, how hepatocytes in distinct anatomic locations within the liver lobule are replenished under homeostasis and injury-induced regeneration remains elusive. Using a newly developed bacterial artificial chromosome (BAC)-transgenic mouse model, we demonstrate that Lgr5 expression in the liver is restricted to a unique subset of hepatocytes most adjacent to the central veins. Genetic lineage tracing revealed that pericentral Lgr5+ hepatocytes have a long lifespan and mainly contribute to their own lineage maintenance during postnatal liver development and homeostasis. Remarkably, these hepatocytes also fuel the regeneration of their own lineage during the massive and rapid regeneration process following two-thirds partial hepatectomy. Moreover, Lgr5+ hepatocytes are found to be the main cellular origin of diethylnitrosamine (DEN)-induced hepatocellular carcinoma (HCC) and are highly susceptible to neoplastic transformation triggered by activation of Erbb pathway. Our findings establish an unexpected self-maintaining mode for a defined subset of hepatocytes during liver homeostasis and regeneration, and identify Lgr5+ pericentral hepatocytes as major cells of origin in HCC development.

RACK1/TRAF2 regulation of modulator of apoptosis-1 (MOAP-1).

MOAP-1 is a pro-apoptotic tumor suppressor molecule with a growing set of known interacting partners. We have demonstrated that during death receptor-dependent apoptosis, MOAP-1 is recruited to TNF-R1 or TRAIL-R1, followed by RASSF1A and Bax association. MOAP-1/Bax association promotes Bax conformational change resulting in the translocation of Bax into the mitochondrial membrane, mitochondrial membrane insertion and dysregulation resulting in several hallmark events that execute apoptosis. Although a role in apoptosis is established, it is currently unknown how MOAP-1 is regulated and how it links to Bax to promote apoptosis. In this study, we demonstrate robust association with RACK1, a versatile scaffolding protein that responds to activation of protein kinase C. Furthermore, we can demonstrate that RACK1 functions to bring the E3 ligase, TRAF2, to MOAP-1 in order to undergo a K63-dependent ubiquitination. Furthermore, RACK1 associates with MOAP-1 via electrostatic associations similar to those observed between MOAP-1/RASSF1A and MOAP-1/TNF-R1. These events illustrate the complex nature of MOAP-1 regulation and characterizes the important role of the scaffolding protein, RACK1, in influencing MOAP-1 biology.

A soluble form of the pilus protein FimA targets the VDAC-hexokinase complex at mitochondria to suppress host cell apoptosis.

Inhibition of apoptotic response of host cells during an early phase of infection is a strategy used by many enteroinvasive bacterial pathogens to enhance their survival. Here, we report the identification of a soluble form of the pilus protein FimA from the culture supernatants of E. coli K1, Salmonella, and Shigella that can potently inhibit Bax-mediated release of cytochrome c from isolated mitochondria. Similar to the infected cells, HCT116 cells stably expressing FimA display a delay in the integration of Bax into outer mitochondrial membrane induced by apoptotic stimuli. FimA targets to mitochondria through binding to VDAC1, which is a prerequisite step for E. coli K1 to render the short-term blockade of apoptotic death in the host cells. Interestingly, FimA strengthens the VDAC1-hexokinase interaction and prevents dissociation of hexokinase from VDAC1 triggered by apoptotic stimuli. Together, these data thus reveal a paradigm of antiapoptosis mechanism undertaken by the enteroinvasive bacteria.

Baxbeta: a constitutively active human Bax isoform that is under tight regulatory control by the proteasomal degradation mechanism.

Although mRNAs of multiple isoforms of Bax, which encodes a central regulator of apoptosis signaling, have been reported, only Baxalpha protein has been well documented and studied. Baxalpha exists in latent form and is activated upon apoptosis induction through conformational changes. Here we demonstrate that Baxbeta protein is ubiquitously present among human cells, but its activity is restricted through stringent regulation by proteasomal degradation. In contrast to Baxalpha, native Baxbeta spontaneously integrates into mitochondrial membrane and is highly potent in inducing cytochrome c release from mitochondria. Remarkably, Baxbeta protein is upregulated by apoptotic stimuli via inhibition of its ubiquitination process, and stable expression of Baxbeta in HCT116-Bax(-/-) cells restores their sensitivity to multiple stimuli. Baxbeta associates with and promotes Baxalpha activation. Moreover, selective knockdown of Baxbeta desensitizes HCT116-Bax(+/-) cells to Bax-dependent apoptosis signaling. These observations underscore the plasticity of human Bax in serving its role as a "gatekeeper" for apoptosis.

Modulator of apoptosis 1 (MOAP-1) is a tumor suppressor protein linked to the RASSF1A protein.

Modulator of apoptosis 1 (MOAP-1) is a BH3-like protein that plays key roles in cell death or apoptosis. It is an integral partner to the tumor suppressor protein, Ras association domain family 1A (RASSF1A), and functions to activate the Bcl-2 family pro-apoptotic protein Bax. Although RASSF1A is now considered a bona fide tumor suppressor protein, the role of MOAP-1 as a tumor suppressor protein has yet to be determined. In this study, we present several lines of evidence from cancer databases, immunoblotting of cancer cells, proliferation, and xenograft assays as well as DNA microarray analysis to demonstrate the role of MOAP-1 as a tumor suppressor protein. Frequent loss of MOAP-1 expression, in at least some cancers, appears to be attributed to mRNA down-regulation and the rapid proteasomal degradation of MOAP-1 that could be reversed utilizing the proteasome inhibitor MG132. Overexpression of MOAP-1 in several cancer cell lines resulted in reduced tumorigenesis and up-regulation of genes involved in cancer regulatory pathways that include apoptosis (p53, Fas, and MST1), DNA damage control (poly(ADP)-ribose polymerase and ataxia telangiectasia mutated), those within the cell metabolism (IR-α, IR-ß, and AMP-activated protein kinase), and a stabilizing effect on microtubules. The loss of RASSF1A (an upstream regulator of MOAP-1) is one of the earliest detectable epigenetically silenced tumor suppressor proteins in cancer, and we speculate that the additional loss of function of MOAP-1 may be a second hit to functionally compromise the RASSF1A/MOAP-1 death receptor-dependent pathway and drive tumorigenesis.

TRIM39 regulates cell cycle progression and DNA damage responses via stabilizing p21.

The biological function of Tripartite Motif 39 (TRIM39) remains largely unknown. In this study, we report that TRIM39 regulates the steady-state levels of p21 and is a pivotal determinant of cell fate. Ablation of TRIM39 leads to destabilization of p21 and increased G1/S transition in unperturbed cells. Furthermore, DNA damage-induced p21 accumulation is completely abolished in cells with depleted TRIM39. As a result, silencing of TRIM39 abrogates the G2 checkpoint induced by genotoxic stress, leading to increased mitotic entry and, ultimately, apoptosis. Importantly, we show p21 is a crucial downstream effector of TRIM39 mediating G1/S transition and DNA damage-induced G2 arrest. Mechanistically, TRIM39 interacts with p21, which subsequently prevents Cdt2 from binding to p21, therefore blocking ubiquitylation and proteasomal degradation of p21 mediated by CRL4(Cdt2) E3 ligase. Strikingly, we found a significant correlation between p21 abundance and TRIM39 expression levels in human hepatocellular carcinoma samples. Our findings identify a causal role for TRIM39 in regulating cell cycle progression and the balance between cytostasis and apoptosis after DNA damage via stabilizing p21.

RNF13, a RING finger protein, mediates endoplasmic reticulum stress-induced apoptosis through the inositol-requiring enzyme (IRE1α)/c-Jun NH2-terminal kinase pathway.

Disturbance of homeostasis at endoplasmic reticulum (ER) causes stress to cells that in turn triggers an adaptive signaling pathway termed unfolded protein response for the purpose of restoring normal cellular physiology or initiating signaling events leading to apoptosis. Identification of those genes that are involved in the unfolded protein response-mediated apoptotic signaling pathway would be valuable toward elucidating the molecular mechanism underlying the relationship between ER stress and apoptosis. We initiated a genetic screen by using the retroviral insertion mutation system to search for genes whose inactivation confers resistance to apoptosis induction by staurosporine. Using this approach, RING finger protein 13 (RNF13) was identified. Interestingly, RNF13 is highly enriched in ER. RNF13 knockdown cells are resistant to apoptosis and JNK activation triggered by ER stress. Conversely, overexpression of RNF13 induces JNK activation and caspase-dependent apoptosis. The RING and transmembrane domains of RNF13 are both required for its effects on JNK activation and apoptosis. Moreover, systematic analysis of the involvement of individual signaling components in the ER stress pathway using knockdown approach reveals that RNF13 acts upstream of the IRE1α-TRAF2 signaling axis for JNK activation and apoptosis. Finally, RNF13 co-immunoprecipitates with IRE1α, and the intact RING domain is also required for mediating its interaction. Together, our data support a model that RNF13 is a critical mediator for facilitating ER stress-induced apoptosis through the activation of the IRE1α-TRAF2-JNK signaling pathway.

p62/SQSTM1 in liver diseases: the usual suspect with multifarious identities.

p62/Sequestosome-1 (SQSTM1) is a selective autophagy receptor that recruits and delivers intracellular substrates for bulk clearance through the autophagy lysosomal pathway. Interestingly, p62 also serves as a signaling scaffold to participate in the regulation of multiple physiological processes, including oxidative stress response, metabolism, inflammation, and programmed cell death. Perturbation of p62 activity has been frequently found to be associated with the pathogenesis of many liver diseases. p62 has been identified as a critical component of protein aggregates in the forms of Mallory-Denk bodies (MDBs) or intracellular hyaline bodies (IHBs), which are known to be frequently detected in biopsy samples from alcoholic steatohepatitis (ASH), non-alcoholic steatohepatitis (NASH), and hepatocellular carcinoma (HCC) patients. Importantly, abundance of these p62 inclusion bodies is increasingly recognized as a biomarker for NASH and HCC. Although the level of p62 bodies seems to predict the progression and prognosis of these liver diseases, understanding of the underlying mechanisms by which p62 regulates and contributes to the development and progression of these diseases remains incomplete. In this review, we will focus on the function and regulation of p62, and its pathophysiological roles in the liver, by critically reviewing the findings from preclinical models that recapitulate the pathogenesis and manifestation of these liver diseases in humans. In addition, we will also explore the suitability of p62 as a predictive biomarker and a potential therapeutic target for the treatment of liver diseases, including NASH and HCC, as well as recent development of small-molecule compounds for targeting the p62 signaling axis.

Trajectory of immune evasion and cancer progression in hepatocellular carcinoma.

Immune evasion is key to cancer initiation and later at metastasis, but its dynamics at intermediate stages, where potential therapeutic interventions could be applied, is undefined. Here we show, using multi-dimensional analyses of resected tumours, their adjacent non-tumour tissues and peripheral blood, that extensive immune remodelling takes place in patients with stage I to III hepatocellular carcinoma (HCC). We demonstrate the depletion of anti-tumoural immune subsets and accumulation of immunosuppressive or exhausted subsets along with reduced tumour infiltration of CD8 T cells peaking at stage II tumours. Corresponding transcriptomic modification occur in the genes related to antigen presentation, immune responses, and chemotaxis. The progressive immune evasion is validated in a murine model of HCC. Our results show evidence of ongoing tumour-immune co-evolution during HCC progression and offer insights into potential interventions to reverse, prevent or limit the progression of the disease.

Mitochondrial translocation of cofilin is an early step in apoptosis induction.

Increasing evidence suggests that movement of key proteins in or out of mitochondria during apoptosis is essential for the regulation of apoptosis. Here, we report identification of the actin-binding protein cofilin by a proteomic approach, as such a factor translocated from cytosol into mitochondria after induction of apoptosis. We found that after induction of apoptosis, cofilin was translocated to mitochondria before release of cytochrome c. Reduction of cofilin protein levels with small-interfering RNA (siRNA) resulted in inhibition of both cytochrome c release and apoptosis. Only dephosphorylated cofilin was translocated to mitochondria, and the cofilin S3D mutant, which mimicks the phosphorylated form, suppressed mitochondrial translocation and apoptosis. Translocation was achieved through exposure of an amino-terminal mitochondrial targeting signal in combination with carboxy-terminal sequences. When correctly targeted to mitochondria, cofilin induced massive apoptosis. The apoptosis-inducing ability of cofilin, but not its mitochondrial localization, was dependent on the functional actin-binding domain. Thus, domains involved in mitochondrial targeting and actin binding are indispensable for its pro-apoptotic function. Our data suggest that cofilin has an important function during the initiation phase of apoptosis.

The BAX-binding protein MOAP1 associates with LC3 and promotes closure of the phagophore.

MOAP1 (modulator of apoptosis 1) is a BAX-binding protein tightly regulated by the ubiquitin-proteasome system. Apoptotic stimuli stabilize MOAP1 protein and facilitate its interaction with BAX to promote apoptosis. Here we show that in contrast to being resistant to apoptotic stimuli, MOAP1-deficient cells are hypersensitive to cell death mediated by starvation rendered by EBSS treatment. MOAP1-deficient cells exhibited impairment in macroautophagy/autophagy signaling induced by EBSS. Mechanistic analysis revealed that MOAP1-deficient cells had no notable defect in the recruitment of the pre-autophagosomal phosphatidylinositol-3-phosphate (PtdIns3P)-binding proteins, ZFYVE1/DFCP1 and WIPI2, nor in the LC3 lipidation mechanism regulated by the ATG12-ATG5-ATG16L1 complex upon EBSS treatment. Interestingly, MOAP1 is required for facilitating efficient closure of phagophore in the EBSS-treated cells. Analysis of LC3-positive membrane structures using Halo-tagged LC3 autophagosome completion assay showed that predominantly unclosed phagophore rather than closed autophagosome was present in the EBSS-treated MOAP1-deficient cells. The autophagy substrate SQSTM1/p62, which is normally contained within the enclosed autophagosome under EBSS condition, was also highly sensitive to degradation by proteinase K in the absence of MOAP1. MOAP1 binds LC3 and the binding is critically dependent on a LC3-interacting region (LIR) motif detected at its N-terminal region. Re-expression of MOAP1, but not its LC3-binding defective mutant, MOAP1-LIR, in the MOAP1-deficient cells, restored EBSS-induced autophagy. Together, these observations suggest that MOAP1 serves a distinct role in facilitating autophagy through interacting with LC3 to promote efficient phagophore closure during starvation.Abbreviations: CQ: Chloroquine; EBSS: Earle's Balanced Salt Solution; GABARAP: Gamma-Amino Butyric Acid Receptor Associated Protein; IF: Immunofluorescence; IP: Immunoprecipitation; LAMP1: Lysosomal-Associated Membrane Protein 1; LIR: LC3-Interacting Region; MAP1LC3/LC3: Microtubule Associated Protein 1 Light Chain 3; MEF: Mouse Embryonic Fibroblast; MOAP1: Modulator of Apoptosis 1; PE: Phosphatidylethanolamine; PtdIns3K: class III PtdIns3K complex I; PtdIns3P: Phosphatidylinositol-3-phosphate; STX17: Syntaxin 17; ULK1: unc-51 like autophagy activating kinase 1.

The hepatitis C virus core protein contains a BH3 domain that regulates apoptosis through specific interaction with human Mcl-1.

The hepatitis C virus (HCV) core protein is known to modulate apoptosis and contribute to viral replication and pathogenesis. In this study, we have identified a Bcl-2 homology 3 (BH3) domain in the core protein that is essential for its proapoptotic property. Coimmunoprecipitation experiments showed that the core protein interacts specifically with the human myeloid cell factor 1 (Mcl-1), a prosurvival member of the Bcl-2 family, but not with other prosurvival members (Bcl-X(L) and Bcl-w). Moreover, the overexpression of Mcl-1 protects against core-induced apoptosis. By using peptide mimetics, core was found to release cytochrome c from isolated mitochondria when complemented with Bad. Thus, core is a bona fide BH3-only protein having properties similar to those of Noxa, a BH3-only member of the Bcl-2 family that binds preferentially to Mcl-1. There are three critical hydrophobic residues in the BH3 domain of the core protein, and they are essential for the proapoptotic property of the core protein. Furthermore, the genotype 1b core protein is more effective than the genotype 2a core protein in inducing apoptosis due to a single-amino-acid difference at one of these hydrophobic residues (residue 119). Replacing this residue in the J6/JFH-1 infectious clone (genotype 2a) with the corresponding amino acid in the genotype 1b core protein produced a mutant virus, J6/JFH-1(V119L), which induced significantly higher levels of apoptosis in the infected cells than the parental J6/JFH-1 virus. Furthermore, the core protein of J6/JFH-1(V119L), but not that of J6/JFH-1, interacted with Mcl-1 in virus-infected cells. Taken together, the core protein is a novel BH3-only viral homologue that contributes to the induction of apoptosis during HCV infection.

TRIM39 is a MOAP-1-binding protein that stabilizes MOAP-1 through inhibition of its poly-ubiquitination process.

Bax, a multi-domain pro-apoptotic Bcl-2 family member, is a key regulator for the release of apoptogenic factors from mitochondria. MOAP-1, which was first isolated from a screen for Bax-associating proteins, interacts with Bax upon apoptotic induction. MOAP-1 is a short-lived protein that is constitutively degraded by the ubiquitin-proteasome system. Apoptotic stimuli upregulate MOAP-1 rapidly through inhibition of its poly-ubiquitination process. However, cellular factors that regulate the stability of MOAP-1 have not yet been identified. In this study, we report the identification of TRIM39 as a MOAP-1-binding protein. TRIM39 belongs to a family of proteins characterized by a Tripartite Motif (TRIM), consisting of RING domain, B-box and coiled-coil domain. Several TRIM family members are known to demonstrate E3 ubiquitin ligase activity. Surprisingly, TRIM39 significantly extends the half-life of MOAP-1 by inhibiting its poly-ubiquitination process. In agreement with its effect on enhancing MOAP-1 stability, TRIM39 sensitizes cells to etoposide-induced apoptosis. Conversely, knockdown of TRIM39 reduces the sensitivity of cells to etoposide-stimulated apoptosis. Furthermore, TRIM39 elevates the level of MOAP-1 in mitochondria and promotes cytochrome c release from isolated mitochondria stimulated by recombinant Bax. Together, these data suggest that TRIM39 can promote apoptosis signalling through stabilization of MOAP-1.

Absence of Stress Response in Dorsal Raphe Nucleus in Modulator of Apoptosis 1-Deficient Mice.

Modulator of apoptosis 1 (MOAP-1) is a Bcl-2-associated X Protein (BAX)-associating protein that plays an important role in regulating apoptosis. It is highly enriched in the brain but its function in this organ remains unknown. Studies on BAX-/- mice suggested that disruption of programmed cell death may lead to abnormal emotional states. We thus hypothesize that MOAP-1-/- mice may also display stress-related behavioral differences and perhaps involved in stress responses in the brain and investigated if a depression-like trait exists in MOAP-1-/- mice, and if so, whether it is age related, and how it relates to central serotonergic stress response in the dorsal raphe nucleus. Young MOAP-1-/- mice exhibit depression-like behavior, in the form of increased immobility time when compared to age-matched wild-type mice in the forced swimming test, which is abolished by acute treatment of fluoxetine. This is supported by data from the tail suspension and sucrose preference tests. Repeated forced swimming stress causes an up-regulation of tryptophan hydroxylase 2 (TPH2) and a down-regulation of brain-derived neurotrophic factor (BDNF) in the dorsal raphe nucleus (DRN) in young wild-type (WT) control mice. In contrast, TPH2 up-regulation was not observed in aged WT mice. Interestingly, such a stress response appears absent in both young and aged MOAP-1-/- mice. Aged MOAP-1-/- and WT mice also have similar immobility times on the forced swimming test. These data suggest that MOAP-1 is required in the regulation of stress response in the DRN. Crosstalk between BDNF and 5-HT appears to play an important role in this stress response.

Interaction of kendomycin and semi-synthetic analogues with the anti-apoptotic protein Bcl-xl.

The cytotoxic macrolide kendomycin was identified as a ligand of Bcl-xl, an anti-apoptotic member of the Bcl-2 protein family. Hydrolysis-stable and protonable semi-synthetic analogues have been obtained that retain cytotoxicity and Bcl-xl binding.

Chelerythrine and sanguinarine dock at distinct sites on BclXL that are not the classic BH3 binding cleft.

The ratio of the levels of pro-survival and pro-apoptotic members of the Bcl-2 protein family is thought to be an important regulatory factor for determining the sensitivity of the mammalian cells to apoptotic stimuli. High levels of expression of pro-survival members such as Bcl(XL) in human cancers were frequently found to be a good prognostic indicator predicting poor response to chemotherapy. The pro-survival members of the Bcl-2 family mediate their effects through heterodimerization with the BH3 region of the pro-apoptotic members. Structural analyses of the binding complex of the BH3 peptide and Bcl(XL) showed that a hydrophobic groove termed the BH3 binding cleft is the docking site for the BH3 region. Chemical mimetics of the BH3 region such as BH3I-1 that target the BH3 binding cleft indeed exhibit pro-apoptotic activities. Chelerythrine (CHE) and sanguinarine (SAN) are natural benzophenanthridine alkaloids that are structurally homologous to each other. CHE was previously identified as an inhibitor of Bcl(XL) function from a high-throughput screen of natural products, but its mode of interaction with Bcl(XL) is not known. By determining the effect of site-directed mutagenesis on ligand binding and using saturation transfer difference (STD) NMR experiments, we have verified locations of these docked ligands. Surprisingly, CHE and SAN bind separately at the BH groove and BH1 region of Bcl(XL) respectively, different from the BH3 binding cleft where other known inhibitors of Bcl(XL) target. Interestingly, certain residues on the flexible loop between helices alpha1 and alpha2 of Bcl(XL) are also perturbed upon CHE, but not SAN or BH3I-1 binding. Although CHE and SAN are similarly effective as BH3I-1 in displacing bound BH3 peptide, they are much more effective in inducing apoptosis, raising the possibility that CHE and SAN might be able to antagonize other pro-survival mechanisms in addition to the one that involves BH3 region binding.

MOAP-1 Mediates Fas-Induced Apoptosis in Liver by Facilitating tBid Recruitment to Mitochondria.

Fas apoptotic signaling regulates diverse physiological processes. Acute activation of Fas signaling triggers massive apoptosis in liver. Upon Fas receptor stimulation, the BH3-only protein Bid is cleaved into the active form, tBid. Subsequent tBid recruitment to mitochondria, which is facilitated by its receptor MTCH2 at the outer mitochondrial membrane (OMM), is a critical step for commitment to apoptosis via the effector proteins Bax or Bak. MOAP-1 is a Bax-binding protein enriched at the OMM. Here, we show that MOAP-1-deficient mice are resistant to Fas-induced hepatocellular apoptosis and lethality. In the absence of MOAP-1, mitochondrial accumulation of tBid is markedly impaired. MOAP-1 binds to MTCH2, and this interaction appears necessary for MTCH2 to engage tBid. These findings reveal a role for MOAP-1 in Fas signaling in the liver by promoting MTCH2-mediated tBid recruitment to mitochondria.

Thioredoxin-dependent regulation of AIF-mediated DNA damage.

The thioredoxin (Trx) system is one major redox system in mammalian cells. One of its component, Trx, is involved in redox homeostasis and many cellular biological processes through participating in disulfide reduction, S-nitrosylation/S-denitrosylation reactions and protein-protein interactions. In this study, we report the identification of a novel interaction between cytosolic/nuclear Trx1 and apoptosis inducing factor (AIF), and the redox sensitivity and biological significance of the Trx-AIF interaction was characterized. Cytosolic Trx1 but not mitochondrial Trx2 was observed to interact with AIF under physiological conditions and Trx1's active site cysteines were crucial for the interaction. Under oxidative stress conditions, Trx-AIF interaction was disrupted. When the treated cells were allowed to recover from oxidative stress by means of removal of the oxidants, interaction between Trx1 and AIF was re-established time-dependently, which underpins the biological relevance of a Trx-dependent redox regulation of AIF-mediated cell death. Indeed, in times of oxidative stress, nuclear translocation of AIF was found to occur concurrently with perturbations to the Trx-AIF interaction. Once localized in the nucleus, reduced Trx1 hindered the interaction between AIF and DNA, thereby bringing about an attenuation of AIF-mediated DNA damage. In conclusion, characterization of the Trx-AIF interaction has led to an understanding of the effect of reduced Trx1 on possibly regulating AIF-dependent cell death through impeding AIF-mediated DNA damage. Importantly, identification of the novel interaction between Trx1 and AIF has provided opportunities to design and develop therapeutically relevant strategies that either promote or prevent this protein-protein interaction for the treatment of different disease states.

The tumor suppressor gene, RASSF1A, is essential for protection against inflammation -induced injury.

Ras association domain family protein 1A (RASSF1A) is a tumor suppressor gene silenced in cancer. Here we report that RASSF1A is a novel regulator of intestinal inflammation as Rassf1a(+/-) , Rassf1a(-/-) and an intestinal epithelial cell specific knockout mouse (Rassf1a (IEC-KO) ) rapidly became sick following dextran sulphate sodium (DSS) administration, a chemical inducer of colitis. Rassf1a knockout mice displayed clinical symptoms of inflammatory bowel disease including: increased intestinal permeability, enhanced cytokine/chemokine production, elevated nuclear factor of kappa light polypeptide gene enhancer in B-cells (NFκB) activity, elevated colonic cell death and epithelial cell injury. Furthermore, epithelial restitution/repair was inhibited in DSS-treated Rassf1a(-/-) mice with reduction of several makers of proliferation including Yes associated protein (YAP)-driven proliferation. Surprisingly, tyrosine phosphorylation of YAP was detected which coincided with increased nuclear p73 association, Bax-driven epithelial cell death and p53 accumulation resulting in enhanced apoptosis and poor survival of DSS-treated Rassf1a knockout mice. We can inhibit these events and promote the survival of DSS-treated Rassf1a knockout mice with intraperitoneal injection of the c-Abl and c-Abl related protein tyrosine kinase inhibitor, imatinib/gleevec. However, p53 accumulation was not inhibited by imatinib/gleevec in the Rassf1a(-/-) background which revealed the importance of p53-dependent cell death during intestinal inflammation. These observations suggest that tyrosine phosphorylation of YAP (to drive p73 association and up-regulation of pro-apoptotic genes such as Bax) and accumulation of p53 are consequences of inflammation-induced injury in DSS-treated Rassf1a(-/-) mice. Mechanistically, we can detect robust associations of RASSF1A with membrane proximal Toll-like receptor (TLR) components to suggest that RASSF1A may function to interfere and restrict TLR-driven activation of NFκB. Failure to restrict NFκB resulted in the inflammation-induced DNA damage driven tyrosine phosphorylation of YAP, subsequent p53 accumulation and loss of intestinal epithelial homeostasis.