Optineurin promotes autophagosome formation by recruiting the autophagy-related Atg12-5-16L1 complex to phagophores containing the Wipi2 protein.

Autophagy is a quality-control mechanism that helps to maintain cellular homeostasis by removing damaged proteins and organelles through lysosomal degradation. During autophagy, signaling events lead to the formation of a cup-shaped structure called the phagophore that matures into the autophagosome. Recruitment of the autophagy-associated Atg12-5-16L1 complex to Wipi2-positive phagophores is crucial for producing microtubule-associated protein 1 light chain 3-II (LC3-II), which is required for autophagosome formation. Here, we explored the role of the autophagy receptor optineurin (Optn) in autophagosome formation. Fibroblasts from Optn knock-out mouse showed reduced LC3-II formation and a lower number of autophagosomes and autolysosomes during both basal and starvation-induced autophagy. However, the number of Wipi2-positive phagophores was not decreased in Optn-deficient cells. We also found that the number of Atg12/16L1-positive puncta and recruitment of the Atg12-5-16L1 complex to Wipi2-positive puncta are reduced in Optn-deficient cells. Of note, Optn was recruited to Atg12-5-16L1-positive puncta, and interacted with Atg5 and also with Atg12-5 conjugate. A disease-associated Optn mutant, E478G, defective in ubiquitin binding, was also defective in autophagosome formation and recruitment to the Atg12-5-16L1-positive puncta. Moreover, we noted that Optn phosphorylation at Ser-177 was required for autophagosome formation but not for Optn recruitment to the phagophore. These results suggest that Optn potentiates LC3-II production and maturation of the phagophore into the autophagosome, by facilitating the recruitment of the Atg12-5-16L1 complex to Wipi2-positive phagophores.

IRF-1-binding site in the first intron mediates interferon-γ-induced optineurin promoter activation.

Optineurin is an adaptor protein involved in signal transduction, membrane vesicle trafficking and autophagy. Optineurin expression is induced by cytokines. Previously we have shown that tumor necrosis factor-α activates optineurin promoter through NF-κB-binding site in the core promoter. However, this promoter was not activated by interferon-γ. Here, we report identification of a functional IRF-1-binding site in the first intron of human optineurin gene that mediates interferon-γ-induced activation of the promoter. Optineurin promoter, containing the contiguous intronic sequences with IRF-1 responsive sites, is strongly activated by IRF-1. Mutational inactivation of IRF-1 site resulted in loss of activation of the promoter by interferon-γ and also by IRF-1. We also show that IRF-1 cooperates with NF-κB to activate optineurin promoter. The synergistic effect of these two transcription factors (IRF-1 and NF-κB) may be involved in cooperative induction of optineurin promoter by interferon-γ and tumor necrosis factor-α.

M98K-OPTN induces transferrin receptor degradation and RAB12-mediated autophagic death in retinal ganglion cells.

Mutations in the autophagy receptor OPTN/optineurin are associated with the pathogenesis of glaucoma and amyotrophic lateral sclerosis, but the underlying molecular basis is poorly understood. The OPTN variant, M98K has been described as a risk factor for normal tension glaucoma in some ethnic groups. Here, we examined the consequence of the M98K mutation in affecting cellular functions of OPTN. Overexpression of M98K-OPTN induced death of retinal ganglion cells (RGC-5 cell line), but not of other neuronal and non-neuronal cells. Enhanced levels of the autophagy marker, LC3-II, a post-translationally modified form of LC3, in M98K-OPTN-expressing cells and the inability of an LC3-binding-defective M98K variant of OPTN to induce cell death, suggested that autophagy contributes to cell death. Knockdown of Atg5 reduced M98K-induced death of RGC-5 cells, further supporting the involvement of autophagy. Overexpression of M98K-OPTN enhanced autophagosome formation and potentiated the delivery of transferrin receptor to autophagosomes for degradation resulting in reduced cellular transferrin receptor levels. Coexpression of transferrin receptor or supplementation of media with an iron donor reduced M98K-induced cell death. OPTN complexes with RAB12, a GTPase involved in vesicle trafficking, and M98K variant shows enhanced colocalization with RAB12. Knockdown of Rab12 increased transferrin receptor level and reduced M98K-induced cell death. RAB12 is present in autophagosomes and knockdown of Rab12 resulted in reduced formation of autolysosomes during starvation-induced autophagy, implicating a role for RAB12 in autophagy. These results also show that transferrin receptor degradation and autophagy play a crucial role in RGC-5 cell death induced by M98K variant of OPTN.

Optineurin mediates a negative regulation of Rab8 by the GTPase-activating protein TBC1D17.

Rab GTPases regulate various membrane trafficking pathways but the mechanisms by which GTPase-activating proteins recognise specific Rabs are not clear. Rab8 is involved in controlling several trafficking processes, including the trafficking of transferrin receptor from the early endosome to the recycling endosome. Here, we provide evidence to show that TBC1D17, a Rab GTPase-activating protein, through its catalytic activity, regulates Rab8-mediated endocytic trafficking of transferrin receptor. Optineurin, a Rab8-binding effector protein, mediates the interaction and colocalisation of TBC1D17 with Rab8. A non-catalytic region of TBC1D17 is required for direct interaction with optineurin. Co-expression of Rab8, but not other Rabs tested, rescues the inhibition of transferrin receptor trafficking by TBC1D17. The activated GTP-bound form of Rab8 is localised to the tubules emanating from the endocytic recycling compartment. Through its catalytic activity, TBC1D17 inhibits recruitment of Rab8 to the tubules and reduces colocalisation of transferrin receptor and Rab8. Knockdown of optineurin or TBC1D17 results in enhanced recruitment of Rab8 to the tubules. A glaucoma-associated mutant of optineurin, E50K, causes enhanced inhibition of Rab8 by TBC1D17, resulting in defective endocytic recycling of transferrin receptor. Our results show that TBC1D17, through its interaction with optineurin, regulates Rab8-mediated endocytic recycling of transferrin receptor and recruitment of Rab8 to the endocytic recycling tubules. We describe a mechanism of regulating a Rab GTPase by an effector protein (optineurin) that acts as an adaptor to bring together a Rab (Rab8) and its GTPase-activating protein (TBC1D17).

NF-kappaB mediates tumor necrosis factor alpha-induced expression of optineurin, a negative regulator of NF-kappaB.

Optineurin is a ubiquitously expressed multifunctional cytoplasmic protein encoded by OPTN gene. The expression of optineurin is induced by various cytokines. Here we have investigated the molecular mechanisms which regulate optineurin gene expression and the relationship between optineurin and nuclear factor kappaB (NF-kappaB). We cloned and characterized human optineurin promoter. Optineurin promoter was activated upon treatment of HeLa and A549 cells with tumor necrosis factor alpha (TNFalpha). Mutation of a putative NF-kappaB-binding site present in the core promoter resulted in loss of basal as well as TNFalpha-induced activity. Overexpression of p65 subunit of NF-kappaB activated this promoter through NF-kappaB site. Oligonucleotides corresponding to this putative NF-kappaB-binding site showed binding to NF-kappaB. TNFalpha-induced optineurin promoter activity was inhibited by expression of inhibitor of NF-kappaB (IkappaBalpha) super-repressor. Blocking of NF-kappaB activation resulted in inhibition of TNFalpha-induced optineurin gene expression. Overexpressed optineurin partly inhibited TNFalpha-induced NF-kappaB activation in Hela cells. Downregulation of optineurin by shRNA resulted in an increase in TNFalpha-induced as well as basal NF-kappaB activity. These results show that optineurin promoter activity and gene expression are regulated by NF-kappaB pathway in response to TNFalpha. In addition these results suggest that there is a negative feedback loop in which TNFalpha-induced NF-kappaB activity mediates expression of optineurin, which itself functions as a negative regulator of NF-kappaB.

Significance of glycosylation patterns of rat Zajdela ascitic hepatocellular carcinoma: effect of enzymatic removal of surface sialic acid on humoral response.

BACKGROUND AND AIM: Rat Zajdela ascitic hepatocellular carcinoma (ZAH) is a malignant cell type with some properties in common with rat hepatocytes. The aim of this study was to determine the glycosylation patterns of surface glycoproteins of two cell lines C and D of ZAH by lectin binding for identification and characterization of tumor-specific markers. METHODS: Plasma membrane proteins from these cells were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, and probed using radio-iodinated lectins. RESULTS: We observed a decrease in the binding of concavalin A, and an increase in the binding of wheat germ agglutinin (WGA) to glycoproteins from the tumor cell lines as compared with those from normal liver cells. We showed that the increase in binding of WGA was mainly due to increased sialylation of the surface glycoproteins of the tumor cells. The major sialylated glycoproteins of the tumor cells contained O-linked carbohydrate chains. It was also shown that removal of surface sialic acid by neuraminidase significantly decreased the lethality of the tumor and led to increased survival of tumor-bearing animals. The decreased lethality of the tumor appears to be due to increased antigenicity of the desialylated tumor cells. CONCLUSION: Taken together, the presence of highly sialylated O-linked glycosylation of gp120 in ZAH tumor cells and its absence in normal liver cells is of significance with respect to the biological properties of this tumor.

Sp1-like sequences mediate human caspase-3 promoter activation by p73 and cisplatin.

Caspase-3 is a cysteine protease that plays a central role in the execution of apoptosis induced by a wide variety of stimuli. However, little is known about the mechanisms involved in the regulation of caspase-3 gene transcription. This study was carried out to characterize the human caspase-3 promoter and to understand the mechanisms involved in the induction of caspase-3 gene expression in response to the anticancer drug cisplatin and p73. Caspase-3 gene expression was induced by treatment of cells with cisplatin, which also induced p73 protein in HeLa and K562 cells. The human caspase-3 promoter was cloned and characterized. p73beta strongly activated the caspase-3 promoter, whereas p73alpha showed less activation. Cisplatin treatment increased caspase-3 promoter activity. Basal and cisplatin-induced promoter activity was inhibited by the p73 inhibitor p73DD. Deletion analysis defined a minimal promoter of 120 base pairs, which showed good basal and p73beta-induced activity. The examination of the minimal promoter sequence showed several putative Sp1 sites, but no p53/p73 site. The caspase-3 promoter was activated by Sp1 in Sp1-deficient Drosophila SL-2 cells. Sp1-induced promoter activity was further enhanced by p73beta in SL-2 cells. Mutation of Sp1 sites in the minimal promoter resulted in a loss of basal and p73-induced promoter activity. These results show that caspase-3 gene transcription is induced by cisplatin, which is mediated partly by p73. We have identified p73 and Sp1 as activators of the caspase-3 promoter. Sp1-like sequences in the minimal promoter not only sustain basal promoter activity, but also mediate p73-induced activation of the promoter.