Impaired STING Activation Due to a Variant in the E3 Ubiquitin Ligase AMFR in a Patient with Severe VZV Infection and Hemophagocytic Lymphohistiocytosis.

Varicella zoster virus (VZV) is a neurotropic alphaherpesvirus exclusively infecting humans, causing two distinct pathologies: varicella (chickenpox) upon primary infection and herpes zoster (shingles) following reactivation. In susceptible individuals, VZV can give rise to more severe clinical manifestations, including disseminated infection, pneumonitis, encephalitis, and vasculopathy with stroke. Here, we describe a 3-year-old boy in whom varicella followed a complicated course with thrombocytopenia, hemorrhagic and necrotic lesions, pneumonitis, and intermittent encephalopathy. Hemophagocytic lymphohistiocytosis (HLH) was strongly suspected and as the condition deteriorated, HLH therapy was initiated. Although the clinical condition improved, longstanding hemophagocytosis followed despite therapy. We found that the patient carries a rare monoallelic variant in autocrine motility factor receptor (AMFR), encoding a ubiquitin ligase involved in innate cytosolic DNA sensing and interferon (IFN) production through the cyclic GMP-AMP synthase-stimulator of IFN genes (cGAS-STING) pathway. Peripheral blood mononuclear cells (PBMCs) from the patient exhibited impaired signaling downstream of STING in response dsDNA and 2'3'-cGAMP, agonists of cGAS and STING, respectively, and fibroblasts from the patient showed impaired type I IFN responses and significantly increased VZV replication. Overexpression of the variant AMFR R594C resulted in decreased K27-linked STING ubiquitination compared to WT AMFR. Moreover, ImageStream technology revealed reduced STING trafficking from ER to Golgi in cells expressing the patient AMFR R594C variant. This was supported by a dose-dependent dominant negative effect of expression of the patient AMFR variant as measured by IFN-ß reporter gene assay. Finally, lentiviral transduction with WT AMFR partially reconstituted 2'3'-cGAMP-induced STING-mediated signaling and ISG expression in patient PBMCs. This work links defective AMFR-STING signaling to severe VZV disease and hyperinflammation and suggests a direct role for cGAS-STING in the control of viral infections in humans. In conclusion, we describe a novel genetic etiology of severe VZV disease in childhood, also representing the first inborn error of immunity related to a defect in the cGAS-STING pathway.

3ß-hydroxysteroid-Δ24 reductase dampens anti-viral innate immune responses by targeting K27 ubiquitination of MAVS and STING.

IMPORTANCE: The precise regulation of the innate immune response is essential for the maintenance of homeostasis. MAVS and STING play key roles in immune signaling pathways activated by RNA and DNA viruses, respectively. Here, we showed that DHCR24 impaired the antiviral response by targeting MAVS and STING. Notably, DHCR24 interacts with MAVS and STING and inhibits TRIM21-triggered K27-linked ubiquitination of MAVS and AMFR-triggered K27-linked ubiquitination of STING, restraining the activation of MAVS and STING, respectively. Together, this study elucidates how one cholesterol key enzyme orchestrates two antiviral signal transduction pathways.

Cholesterol suppresses AMFR-mediated PDL1 ubiquitination and degradation in HCC.

The degradation of proteasomes or lysosomes is emerging as a principal determinant of programmed death ligand 1 (PDL1) expression, which affects the efficacy of immunotherapy in various malignancies. Intracellular cholesterol plays a central role in maintaining the expression of membrane receptors; however, the specific effect of cholesterol on PDL1 expression in cancer cells remains poorly understood. Cholesterol starvation and stimulation were used to modulate the cellular cholesterol levels. Immunohistochemistry and western blotting were used to analyze the protein levels in the samples and cells. Quantitative real-time PCR, co-immunoprecipitation, and confocal co-localization assays were used for mechanistic investigation. A xenograft tumor model was constructed to verify these results in vivo. Our results showed that cholesterol suppressed the ubiquitination and degradation of PDL1 in hepatocellular carcinoma (HCC) cells. Further mechanistic studies revealed that the autocrine motility factor receptor (AMFR) is an E3 ligase that mediated the ubiquitination and degradation of PDL1, which was regulated by the cholesterol/p38 mitogenic activated protein kinase axis. Moreover, lowering cholesterol levels using statins improved the efficacy of programmed death 1 (PD1) inhibition in vivo. Our findings indicate that cholesterol serves as a signal to inhibit AMFR-mediated ubiquitination and degradation of PDL1 and suggest that lowering cholesterol by statins may be a promising combination strategy to improve the efficiency of PD1 inhibition in HCC.

Expression of autocrine motility factor receptor (AMFR) in human breast and lung invasive micropapillary carcinomas.

The aim of this study was to evaluate the clinicopathological significance of autocrine motility factor receptor (AMFR) expression in a variety of human invasive micropapillary carcinomas (IMPC). AMFR expression was compared in 111 samples of a variety of human IMPCs which had intrinsic non-micropapillary components and with 26 cases of control pulmonary adenocarcinoma (CPA, carcinoma without an IMPC component) by immunohistochemistry (IHC). In the 137 cases analysed, AMFR expression was significantly elevated in the IMPC components compared to the non-IMPC components (p = .005) and normal tissues (p < .001). AMFR expression was also higher in the IMPC samples compared to their intrinsic non-IMPC components (p = .0234). Between the 69 cases of lung IMPC and 26 cases of CPA, AMFR expression was notably higher in the IMPC components than in the CPA components (p = .0455). However, there was no significant difference between the non-IMPC components in the lung and the CPA components (p = .4584). Moreover, in breast cancer, elevated AMFR expression was not significantly correlated with mixed type or pure type IMPC (p = .5969) or with age, gender, T stage, or lymph node metastasis (LNM). Between IMPC and CPA of the lung, there was no statistical significance in age, T stage, and LNM, where AMFR expression was higher in IMPC (p = .0071). Thus this study demonstrated that AMFR was overexpressed in a variety of human IMPC components compared with non-micropapillary components. This suggests that AMFR expression is a potential new prognostic indicator for different types of human IMPC, which might thus be a new therapeutic target.

AMFR dysfunction causes autosomal recessive spastic paraplegia in human that is amenable to statin treatment in a preclinical model.

Hereditary spastic paraplegias (HSP) are rare, inherited neurodegenerative or neurodevelopmental disorders that mainly present with lower limb spasticity and muscle weakness due to motor neuron dysfunction. Whole genome sequencing identified bi-allelic truncating variants in AMFR, encoding a RING-H2 finger E3 ubiquitin ligase anchored at the membrane of the endoplasmic reticulum (ER), in two previously genetically unexplained HSP-affected siblings. Subsequently, international collaboration recognized additional HSP-affected individuals with similar bi-allelic truncating AMFR variants, resulting in a cohort of 20 individuals from 8 unrelated, consanguineous families. Variants segregated with a phenotype of mainly pure but also complex HSP consisting of global developmental delay, mild intellectual disability, motor dysfunction, and progressive spasticity. Patient-derived fibroblasts, neural stem cells (NSCs), and in vivo zebrafish modeling were used to investigate pathomechanisms, including initial preclinical therapy assessment. The absence of AMFR disturbs lipid homeostasis, causing lipid droplet accumulation in NSCs and patient-derived fibroblasts which is rescued upon AMFR re-expression. Electron microscopy indicates ER morphology alterations in the absence of AMFR. Similar findings are seen in amfra-/- zebrafish larvae, in addition to altered touch-evoked escape response and defects in motor neuron branching, phenocopying the HSP observed in patients. Interestingly, administration of FDA-approved statins improves touch-evoked escape response and motor neuron branching defects in amfra-/- zebrafish larvae, suggesting potential therapeutic implications. Our genetic and functional studies identify bi-allelic truncating variants in AMFR as a cause of a novel autosomal recessive HSP by altering lipid metabolism, which may potentially be therapeutically modulated using precision medicine with statins.

Human liver cytochrome P450 3A4 ubiquitination: molecular recognition by UBC7-gp78 autocrine motility factor receptor and UbcH5a-CHIP-Hsc70-Hsp40 E2-E3 ubiquitin ligase complexes.

CYP3A4 is an abundant and catalytically dominant human liver endoplasmic reticulum-anchored cytochrome P450 enzyme engaged in the biotransformation of endo- and xenobiotics, including >50% of clinically relevant drugs. Alterations of CYP3A4 protein turnover can influence clinically relevant drug metabolism and bioavailability and drug-drug interactions. This CYP3A4 turnover involves endoplasmic reticulum-associated degradation via the ubiquitin (Ub)-dependent 26 S proteasomal system that relies on two highly complementary E2 Ub-conjugating-E3 Ub-ligase (UBC7-gp78 and UbcH5a-C terminus of Hsc70-interacting protein (CHIP)-Hsc70-Hsp40) complexes, as well as protein kinases (PK) A and C. We have documented that CYP3A4 Ser/Thr phosphorylation (Ser(P)/Thr(P)) by PKA and/or PKC accelerates/enhances its Lys ubiquitination by either of these E2-E3 systems. Intriguingly, CYP3A4 Ser(P)/Thr(P) and ubiquitinated Lys residues reside within the cytosol-accessible surface loop and/or conformationally assembled acidic Asp/Glu clusters, leading us to propose that such post-translational Ser/Thr protein phosphorylation primes CYP3A4 for ubiquitination. Herein, this possibility was examined through various complementary approaches, including site-directed mutagenesis, chemical cross-linking, peptide mapping, and LC-MS/MS analyses. Our findings reveal that such CYP3A4 Asp/Glu/Ser(P)/Thr(P) surface clusters are indeed important for its intermolecular electrostatic interactions with each of these E2-E3 subcomponents. By imparting additional negative charge to these Asp/Glu clusters, such Ser/Thr phosphorylation would generate P450 phosphodegrons for molecular recognition by the E2-E3 complexes, thereby controlling the timing of CYP3A4 ubiquitination and endoplasmic reticulum-associated degradation. Although the importance of phosphodegrons in the CHIP targeting of its substrates is known, to our knowledge this is the first example of phosphodegron involvement in gp78-substrate recruitment, an important step in CYP3A4 proteasomal degradation.

Autocrine motility factor receptor promotes the malignancy of glioblastoma by regulating cell migration and invasion.

OBJECTIVE: One of the important causes of death in cancer patients is malignant metastasis, invasion, and metastasis of tumor cells. Metastasis is also the most basic physiological characteristics and pathogenesis of various tumors. Previously published studies have suggested that autocrine motor factor receptor (AMFR) is the key regulator of tumor cell migration and invasion. Meanwhile, AMFR is highly expressed in esophageal tumors, gastrointestinal tumors, and bladder cancer, and it is also involved in its pathogenesis. However, the role of AMFR in glioblastoma has not been reported. METHODS: In order to study the role of AMFR in the cell migration and invasion of glioblastoma, AMFR was silenced using siRNA and overexpressed using cDNA. Immunoblotting analysis and real-time quantitative polymerase chain reaction (PCR) were employed to assess the expression of AMFR. We conducted wound healing assay, cell migration assay, and tumorsphere formation assay to detect the invasion and metastatic ability of glioblastoma. RESULTS: This study found that the level of AMFR expression was significantly correlated with the malignant degree of glioma tissue in clinic samples. AMFR silencing decreased cell migration and invasion of LN229. Overexpression of AMFR significantly increased cell migration and invasion of U251. CONCLUSION: This study suggests that AMFR could be used as a therapeutic strategy for the clinical treatment of glioblastoma.

AMFR promotes innate immunity activation and proteasomal degradation of HMGCR in response to influenza virus infection in A549 cells.

Differential regulation of the 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), which is considered the rate-limiting enzyme of the cholesterol biosynthesis pathway, has been reported in case of infection with many viruses. In our study, we have found that influenza virus infection decreases total cellular cholesterol level which is directly related to the downregulation of HMGCR protein. We found that HMGCR is degraded through ubiquitination and proteasomal-mediated pathway upon viral infection. Upregulation of Autocrine Motility Factor Receptor (AMFR), which is an E3-ubiquitin ligase of HMGCR, was also observed. Furthermore, knockdown of AMFR inhibits ubiquitination of HMGCR and also leads to inactivation of the innate immunity components TANK-binding kinase 1 (TBK1) and Interferon regulatory factor 3 (IRF3). Our study is the first to show the role of HMGCR and AMFR in influenza virus infection and reveals that AMFR plays a crucial role in the downregulation of HMGCR and the activation of innate immunity following influenza virus infection.

The chemokine CCL1 facilitates pulmonary fibrosis by promoting macrophage migration and M2 polarization.

Macrophage M2 polarization has been identified in the pathogenesis of pulmonary fibrosis (PF), but the mediators that drive the macrophage M2 program in PF need to be clarified. We showed that the expression of AMFR and CCR8, two known receptors of CCL1, was increased in macrophages from lungs of mice with bleomycin (BLM)-induced PF. Deficiency in either AMFR or CCR8 in macrophages protected mice from BLM-induced PF. In vitro experiments revealed that CCL1 recruited macrophages by binding to its classical receptor CCR8 and drove the macrophage M2 phenotype via its interaction with the recently identified receptor AMFR. Mechanistic studies revealed that the CCL1-AMFR interaction enhanced CREB/C/EBPß signaling to promote the macrophage M2 program. Together, our findings reveal that CCL1 acts as a mediator of macrophage M2 polarization and could be a therapeutic target in PF.

AMFR and DCTN2 genes cause transplantation resistance of adipose-derived mesenchymal stem cells in type 1 diabetes mellitus.

Type 1 diabetes mellitus (T1DM) is characterized by pancreatic beta cell destruction by autoantibodies and other factors, resulting in insulin secretion deficiency. Therefore, beta cell regeneration would be necessary to cure the disease. Nevertheless, the impact of type 1 diabetes on the stemness and transplantation efficiency of stem cells has not been previously described. In this study, we used next-generation sequencing to identify genes differentially expressed in T1DM adipose-derived stem cells (T1DM ADSCs) that originate from patients with type 1 diabetes. Furthermore, we evaluated their effects on transplantation efficiency following xenotransplantation into immunodeficient mice. In the T1DM ADSCs transplant group, the volume and weight of the graft were significantly reduced and the transplant efficiency was reduced. Next-generation sequencing and quantitative PCR results showed that T1DM ADSCs had significantly increased expression of AMFR and DCTN2. AMFR and DCTN2 gene knockdown in T1DM ADSC significantly restored cell proliferation and stem cell marker expression. Therefore, transplantation of T1DM ADSCs, in which AMFR and DCTN2 were knocked down, into immunodeficient mice improved transplant efficiency. This study revealed that AMFR and DCTN2 can reduce transplantation efficiency of T1DM ADSCs. Focusing on AMFR and DCTN2 is expected to increase the efficiency of stem cell transplantation therapy for diabetic patients.

RETREG1/FAM134B mediated autophagosomal degradation of AMFR/GP78 and OPA1 -a dual organellar turnover mechanism.

Turnover of cellular organelles, including endoplasmic reticulum (ER) and mitochondria, is orchestrated by an efficient cellular surveillance system. We have identified a mechanism for dual regulation of ER and mitochondria under stress. It is known that AMFR, an ER E3 ligase and ER-associated degradation (ERAD) regulator, degrades outer mitochondrial membrane (OMM) proteins, MFNs (mitofusins), via the proteasome and triggers mitophagy. We show that destabilized mitochondria are almost devoid of the OMM and generate "mitoplasts". This brings the inner mitochondrial membrane (IMM) in the proximity of the ER. When AMFR levels are high and the mitochondria are stressed, the reticulophagy regulatory protein RETREG1 participates in the formation of the mitophagophore by interacting with OPA1. Interestingly, OPA1 and other IMM proteins exhibit similar RETREG1-dependent autophagosomal degradation as AMFR, unlike most of the OMM proteins. The "mitoplasts" generated are degraded by reticulo-mito-phagy - simultaneously affecting dual organelle turnover.Abbreviations: AMFR/GP78: autocrine motility factor receptor; BAPTA: 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; BFP: blue fluorescent protein; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; CNBr: cyanogen bromide; ER: endoplasmic reticulum; ERAD: endoplasmic-reticulum-associated protein degradation; FL: fluorescence, GFP: green fluorescent protein; HA: hemagglutinin; HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; IMM: inner mitochondrial membrane; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MFN: mitofusin, MGRN1: mahogunin ring finger 1; NA: numerical aperature; OMM: outer mitochondrial membrane; OPA1: OPA1 mitochondrial dynamin like GTPase; PRNP/PrP: prion protein; RER: rough endoplasmic reticulum; RETREG1/FAM134B: reticulophagy regulator 1; RFP: red fluorescent protein; RING: really interesting new gene; ROI: region of interest; RTN: reticulon; SEM: standard error of the mean; SER: smooth endoplasmic reticulum; SIM: structured illumination microscopy; SQSTM1/p62: sequestosome 1; STED: stimulated emission depletion; STOML2: stomatin like 2; TOMM20: translocase of outer mitochondrial membrane 20; UPR: unfolded protein response.

Why Hepatic CYP2E1-Elevation by Itself Is Insufficient for Inciting NAFLD/NASH: Inferences from Two Genetic Knockout Mouse Models.

Hepatic cytochrome P450 CYP2E1 is an enzyme engaged in the metabolic biotransformation of various xenobiotics and endobiotics, resulting in both detoxification and/or metabolic activation of its substrates to more therapeutic or toxic products. Elevated hepatic CYP2E1 content is implicated in various metabolic diseases including alcoholic liver disease, nonalcoholic fatty liver disease (NAFLD)/nonalcoholic steatohepatitis (NASH), diabetes and obesity. While hepatic CYP2E1 elevation is considered essential to the pathogenesis of these liver diseases, our findings in two mouse models of E3 ubiquitin ligase genetic ablation fed a regular lab chow diet, argue that it is not sufficient for triggering NAFLD/NASH. Thus, albeit comparable hepatic CYP2E1 elevation and functional stabilization in these two models upon E3 ubiquitin ligase genetic ablation and consequent disruption of its ubiquitin-dependent proteasomal degradation, NAFLD/NASH was only observed in the mouse livers that exhibited concurrent SREBP1c-transcriptional upregulation of hepatic lipogenesis. These findings reinforce the critical complicity of an associated prolipogenic scenario induced by either an inherently upregulated hepatic lipogenesis or a high fat/high carbohydrate diet in CYP2E1-mediated NAFLD/NASH.

Autocrine motility factor and its receptor expression in musculoskeletal tumors.

Management of aggressive malignant musculoskeletal tumors is clinically challenging and awaits the identification of regulator(s) that can be therapeutically used to improve patient outcome. Autocrine motility factor (AMF), a secreted cytokine, is known to alter the bone microenvironment by linking to its receptor AMFR (AMF Receptor), leading to tumor progression. It was noted that both the ligand and its receptor belong to the moonlighting family of proteins, as they contribute to intracellular metabolic function such as glycolysis and gluconeogenesis by expressing glucose-6-phosphate isomerase AMF/GPI and higher protein degradation by expressing AMFR/gp78 functioning as ubiquitin ligase activity. Thus, AMF/GPI and AMFR/gp78 contribute to higher metabolic turnover of protein and glucose. Recently, a large-scale cohort study including 23 different histological types of musculoskeletal tumors revealed that patients with osteosarcoma, multiple myeloma, rhabdomyosarcoma, and angiosarcoma tend to express higher levels of AMF, whereas multiple myeloma patients expressed high levels of AMFR. Consistently, the cellular data showed that a variety of musculoskeletal tumors express AMF and components of bone microenvironment express AMFR. Thus, a novel outlook suggests a cellular link and cross-talk between musculoskeletal tumors and the skeletal milieu are regulated by AMF-AMFR signaling. This review will highlight the pharmacological need for AMF and AMFR inhibitors as unmet medical needs for patients with malignant musculoskeletal tumors.

Amplification of autocrine motility factor and its receptor in multiple myeloma and other musculoskeletal tumors.

Autocrine motility factor (AMF: GPI) and its receptor AMFR (AMF Receptor: gp78) regulate the metastatic process. Here, we have tested the expression levels of AMF, AMFR, and AMF × AMFR in 1348 patients with musculoskeletal tumor. The results depicted here identified that multiple myeloma highly express AMF × AMFR value as compared with normal bone samples (p < 0.00001). To visualize the AMF × AMFR autocrine amplification in multiple myeloma microenvironment, we have developed a novel software aimed at analyzing numerous cell-to-cell and ligand-to-receptor interactions, i.e., Environmentome. It has led to the identification that myeloma-associated interactions with normal bone cells including osteoblast, osteoclast, immunological components, and others in a paracrine manner. In conclusion, the data showed that AMF × AMFR amplification is a clinical manifestation in bone microenvironment of multiple myeloma.

Cytochrome P450 endoplasmic reticulum-associated degradation (ERAD): therapeutic and pathophysiological implications.

The hepatic endoplasmic reticulum (ER)-anchored cytochromes P450 (P450s) are mixed-function oxidases engaged in the biotransformation of physiologically relevant endobiotics as well as of myriad xenobiotics of therapeutic and environmental relevance. P450 ER-content and hence function is regulated by their coordinated hemoprotein syntheses and proteolytic turnover. Such P450 proteolytic turnover occurs through a process known as ER-associated degradation (ERAD) that involves ubiquitin-dependent proteasomal degradation (UPD) and/or autophagic-lysosomal degradation (ALD). Herein, on the basis of available literature reports and our own recent findings of in vitro as well as in vivo experimental studies, we discuss the therapeutic and pathophysiological implications of altered P450 ERAD and its plausible clinical relevance. We specifically (i) describe the P450 ERAD-machinery and how it may be repurposed for the generation of antigenic P450 peptides involved in P450 autoantibody pathogenesis in drug-induced acute hypersensitivity reactions and liver injury, or viral hepatitis; (ii) discuss the relevance of accelerated or disrupted P450-ERAD to the pharmacological and/or toxicological effects of clinically relevant P450 drug substrates; and (iii) detail the pathophysiological consequences of disrupted P450 ERAD, contributing to non-alcoholic fatty liver disease (NAFLD)/non-alcoholic steatohepatitis (NASH) under certain synergistic cellular conditions.

Betulinic Acid Exerts Cytotoxic Activity Against Multidrug-Resistant Tumor Cells via Targeting Autocrine Motility Factor Receptor (AMFR).

Betulinic acid (BetA) is a naturally occurring pentacyclic triterpene isolated from the outer bark of white-barked birch trees and many other medicinal plants. Here, we studied betulinic acid's cytotoxic activity against drug-resistant tumor cell lines. P-glycoprotein (MDR1/ABCB1) and BCRP (ABCG2) are known ATP-binding cassette (ABC) drug transporters that mediating MDR. ABCB5 is a close relative to ABCB1, which also mediates MDR. Constitutive activation of the EGF receptor is tightly linked to the development of chemotherapeutic resistance. BetA inhibited P-gp, BCRP, ABCB5 and mutation activated EGFR overexpressing cells with similar efficacy as their drug-sensitive parental counterparts. Furthermore, the mRNA expressions of ABCB1, BCRP, ABCB5 and EGFR were not related to the 50% inhibition concentrations (IC50) for BetA in a panel of 60 cell lines of the National Cancer Institute (NCI), USA. In addition to well-established MDR mechanisms, we attempted to identify other molecular mechanisms that play a role in mediating BetA's cytotoxic activity. For this reason, we performed COMPARE and hierarchical cluster analyses of the transcriptome-wide microarray-based mRNA expression of the NCI cell lines panel. Various genes significantly correlating to BetA's activity were involved in different biological processes, e.g., cell cycle regulation, microtubule formation, signal transduction, transcriptional regulation, chromatin remodeling, cell adhesion, tumor suppression, ubiquitination and proteasome degradation. Immunoblotting and in silico analyses revealed that the inhibition of AMFR activity might be one of the mechanisms for BetA to overcome MDR phenotypes. In conclusion, BetA may have therapeutic potential for the treatment of refractory tumors.

The E3 ubiquitin ligase gp78 protects against ER stress in zebrafish liver.

Enhanced endoplasmic reticulum (ER)-associated protein degradation (ERAD) activity by the unfolded protein response (UPR) represents one of the mechanisms for restoring ER homeostasis. In vitro evidence indicates that the mammalian gp78 protein is an E3 ubiquitin ligase that facilitates ERAD by polyubiquitinating and targeting proteins for proteasomal degradation under both physiologic and stress conditions. However, the in vivo function of gp78 in maintaining ER protein homeostasis remains untested. Here we show that like its mammalian counterpart, the zebrafish gp78 is also an E3 ubiquitin ligase as revealed by in vitro ubiquitination assays. Expression analysis uncovered that gp78 is highly expressed in several organs, including liver and brain, of both larval and adult fish. Treatment of larvae or adult fish with tunicamycin induces ER stress and upregulates the expression of several key components of the gp78 ERAD complex in the liver. Moreover, liver-specific overexpression of the dominant-negative form of gp78 (gp78-R2M) renders liver more sensitive to tunicamycin-induced ER stress and enhances the expression of sterol response element binding protein (Srebp)-target genes, which was largely suppressed in fish overexpressing wild-type gp78. Together, these data indicate that gp78 plays a critical role in protecting against ER stress in liver.

Immunohistochemical expression of Type IV Collagen and Autocrine Motility Factor Receptor in Odontogenic Tumours.

BACKGROUND: Autocrine motility factor receptor (AMFR) is a tumour motility stimulating protein secreted by tumour cells. The protein encoded by this gene is a glycosylated transmembrane protein and a receptor for autocrine motility factor. It has been known to play a role in progression of neoplastic lesions. Basement membranes are specialized extracellular matrices that serve as structural barriers as well as substrates for cellular interactions. The network of type IV collagen is thought to define the scaffold integrating other components such as laminins and perlecan into highly organized supramolecular architecture. The aim of this study was to determine and evaluate the immunohistochemical expression of Type IV Collagen and Autocrine motility factor receptor in odontogenic lesions. MATERIALS AND METHODS: Immunohistochemical expression of Type IV Collagen and Autocrine motility factor receptor was evaluated in 31 odontogenic lesions, including unicystic ameloblastoma, multicystic ameloblastoma, keratocystic odontogenic tumour and ameloblastic carcinoma. Normal follicular tissue formed the control. RESULTS: Maximum expression for Type IV Collagen was seen in multicystic ameloblastoma and minimum expression in keratocystic odontogenic tumour. The maximum expression of AMFR was seen in ameloblastic carcinoma and minimum expression in multicystic ameloblastoma. CONCLUSION: The results of this study suggested an association of loss of expression of type IV Collagen with progression of lesion. AMFR expression was found to be associated with the aggressive potential of tumours.