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.

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.

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.

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.

Modulator of apoptosis-1 is a potential therapeutic target in acute ischemic injury.

Modulator of apoptosis 1 (MOAP-1) is a Bax-associating protein highly enriched in the brain. In this study, we examined the role of MOAP-1 in promoting ischemic injuries following a stroke by investigating the consequences of MOAP-1 overexpression or deficiency in in vitro and in vivo models of ischemic stroke. MOAP-1 overexpressing SH-SY5Y cells showed significantly lower cell viability following oxygen and glucose deprivation (OGD) treatment when compared to control cells. Consistently, MOAP-1-/- primary cortical neurons were observed to be more resistant against OGD treatment than the MOAP-1+/+ primary neurons. In the mouse transient middle cerebral artery occlusion (tMCAO) model, ischemia triggered MOAP-1/Bax association, suggested activation of the MOAP-1-dependent apoptotic cascade. MOAP-1-/- mice were found to exhibit reduced neuronal loss and smaller infarct volume 24 h after tMCAO when compared to MOAP-1+/+ mice. Correspondingly, MOAP-1-/- mice also showed better integrity of neurological functions as demonstrated by their performance in the rotarod test. Therefore, both in vitro and in vivo data presented strongly support the conclusion that MOAP-1 is an important apoptotic modulator in ischemic injury. These results may suggest that a reduction of MOAP-1 function in the brain could be a potential therapeutic approach in the treatment of acute stroke.

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.

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.

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.

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.

HUWE1 ubiquitylates and degrades the RAC activator TIAM1 promoting cell-cell adhesion disassembly, migration, and invasion.

The E3 ubiquitin ligase HUWE1, deregulated in carcinoma, has been implicated in tumor formation. Here, we uncover a role for HUWE1 in cell migration and invasion through degrading the RAC activator TIAM1, implying an additional function in malignant progression. In MDCKII cells in response to HGF, HUWE1 catalyzes TIAM1 ubiquitylation and degradation predominantly at cell-cell adhesions, facilitating junction disassembly, migration, and invasion. Depleting HUWE1 or mutating the TIAM1 ubiquitylation site prevents TIAM1 degradation, antagonizing scattering, and invasion. Moreover, simultaneous depletion of TIAM1 restores migration and invasion in HUWE1-depleted cells. Significantly, we show that HUWE1 stimulates human lung cancer cell invasion through regulating TIAM1 stability. Finally, we demonstrate that HUWE1 and TIAM1 protein levels are inversely correlated in human lung carcinomas. Thus, we elucidate a critical role for HUWE1 in regulating epithelial cell-cell adhesion and provide additional evidence that ubiquitylation contributes to spatiotemporal control of RAC.

Enhanced detection of ubiquitin isopeptides using reductive methylation.

Identification of ubiquitination (Ub) sites is of great interest due to the critical roles that the modification plays in cellular regulation. Current methods using mass spectrometry rely upon tryptic isopeptide diglycine tag generation followed by database searching. We present a novel approach to ubiquitin detection based upon the dimethyl labeling of isopeptide N-termini glycines. Ubiquitinated proteins were digested with trypsin and the resulting peptide mixture was derivatized using formaldehyde-D2 solution and sodium cyanoborohydride. The dimethylated peptide mixtures were next separated by liquid chromatography and analyzed on a quadrupole-TOF based mass spectrometer. Diagnostic b2' and a1' ions released from the isopeptide N-terminus upon collision-induced dissociation (CID) were used to spectrally improve the identification of ubiquitinated isopeptides. Proof of principle was established by application to a ubiquitinated protein tryptic digest spiked into a six-protein mix digest background. Extracted ion chromatograms of the a1' and b2' diagnostic product ions from the diglycine tag resulted in a significant reduction in signal complexity and demonstrated a selectivity towards the identification of diglycine branched isopeptides. The method was further shown to be capable of identifying diglycine isopeptides resulting from in-gel tryptic digests of ubiquitin enriched material from a His-Ub transfected cell line. We envisage that these ions may be utilized in global ubiquitination studies with post-acquisition MS/MS (or MSe) data interrogation on high resolution hybrid mass spectrometers. ᅟ

A novel approach to the analysis of SUMOylation with the independent use of trypsin and elastase digestion followed by database searching utilising consecutive residue addition to lysine.

RATIONALE: Identification of sites of protein SUMOylation is of great importance due its functional diversity within the cell. To date, most approaches to this problem rely on site-directed mutagenesis and/or highly specialised mass spectrometry approaches. We present a novel alternative approach to the site mapping of SUMOylation using trypsin and elastase digestion, routine mass spectrometry and an unbiased isotag database searching strategy. METHODS: SUMOylated protein samples were digested with a number of enzymes and the resulting peptides separated using liquid chromatography. Analysis was carried out on both linear ion trap Orbitrap and quadrupole-time-of-flight (Q-TOF)-based mass spectrometers equipped with electrospray ionisation. The data files were subsequently searched using the Mascot algorithm with multiple variable tag modifications corresponding to SUMO-derived fragments. The utility of this approach was demonstrated with di-SUMO 2, di-SUMO 3, SUMO 1-RanGap(418-587) 1 and an enriched population of SUMOylated proteins. RESULTS: Unbiased database searches led to the identification of a number of analytically useful isotags ranging in length from two to four residues. Isopeptide fragments were generated including QTGG (di-SUMO-2/3), TGG (di-SUMO-2/3) and GG (SUMO-1). The method was validated by successfully mapping a number of sites of SUMO modification on SUMO-modified proteins enriched from a cell lysate. CONCLUSIONS: This combination of relaxed enzyme specificity, shortened isotag generation and unbiased database searching enabled confident identification of novel analytically useful SUMOylated isopeptides without a requirement for mutagenesis.