Optineurin, a multifunctional protein involved in glaucoma amyotrophic lateral sclerosis and antiviral signalling.

Glaucomas are a heterogeneous group of neurodegenerative eye diseases that cause blindness and are one of the leading causes of blindness worldwide. Loss of vision in glaucoma occurs due to death of retinal ganglion cells (RGCs) in the optic nerve head (Quigley 1999). Some of the genes known to be involved in causing glaucoma in adults are myocilin, optineurin and WDR36 (Stone et al. 1997; Rezaie et al. 2002; Monemi et al. 2005; Ray and Mookherjee 2009). Mutations in the coding region of the gene OPTN, which codes for the protein optineurin, are associated with certain types of glaucoma. In the original study, families affected with normal tension glaucoma (a sub-type of adult onset primary open angle glaucoma) were analysed for mutations in optineurin (Rezaie et al. 2002). Subsequent studies have shown that in sporadic cases of glaucoma mutations in optineurin are rare, accounting for only about 1% of the cases. Almost all the glaucoma-associated mutations in OPTN are single copy alterations. Most of these mutations are missense mutations. One of the glaucoma-associated mutants (E50K) causes death of retinal ganglion cells in vitro as well as in transgenic mice providing support to the suggestion that mutations in optineurin cause glaucoma (Chalasani et al. 2007; Chi et al. 2010). A recent report described that certain mutations in optineurin are the cause of familial amyotrophic lateral sclerosis (ALS) (Maruyama et al. 2010). Like some forms of glaucomas, ALS is an adult onset progressive neurodegenerative disorder whose hallmark is the selective death of motor neurons of primary motor cortex, brainstem, and spinal cord. Mutations in optineurin were found in familial as well as sporadic cases of ALS. Three types of mutations were observed, two of these being homozygous. One of these homozygous mutations was deletion of exon 5, observed in familial ALS, and the other was Q398X nonsense mutation found in both familial as well sporadic cases. A heterozygous missense mutation E478G was observed in familial ALS (Maruyama et al. 2010). What are the functional defects caused by mutations in optineurin? To address this question we need to understand the function of normal optineurin. Optineurin interacts with several proteins which are involved in various functions (table 1, fi gure 1). On the basis of interactions and other experiments various functions have been proposed for optineurin such as regulation of exocytosis and vesicle traffi c from the Golgi to the plasma membrane, organization of the Golgi stacks, regulation of signalling to transcription factor NF-κB, antiviral signalling, metabotropic glutamate receptor signalling and regulation of gene expression (Hattula and Peranen 2000; Anborgh et al. 2005; Weisschuh et al. 2007; Zhu et al. 2007; Chalasani et al. 2009; del Toro et al. 2009; Mankouri et al. 2010). Optineurin is phosphorylated on serine and tyrosine residues and exists as homohexamers (Ying et al. 2010). Several optineurin-interacting proteins such as Rab8, Huntingtin, myosin VI and TBC1D17 are involved in regulating vesicular membrane traffi c in various cells. Knockdown of optineurin affects structure of the Golgi and reduces transport from the Golgi to the plasma membrane (Sahlender et al. 2005). However, there is no report of any disruption of this transport (from the Golgi to the plasma membrane) or exocytosis by any disease-associated mutant although overexpression of the E50K mutant and to a lesser extent of wild-type optineurin causes breakdown of the Golgi (Park et al. 2006). Recently we and others have shown that knockdown of optineurin reduces endocytic traffi cking of transferrin and its receptor (transferrin receptor, TfR) to the recycling endosomes (Nagabhushana et al. 2010; Park et al. 2010). A glaucoma-causing mutant of optineurin (E50K) impairs traffi cking of TfR possibly due to altered interactions with Rab8 and transferrin receptor. This impaired traffi cking results in accumulation of TfR in large vesicular structures formed by E50K leading to lower level of TfR at the cell surface and hence reduced uptake of transferrin by the E50K expressing cells. It was suggested that impaired traffi cking caused by the E50K mutant might be the cause of cell death induced by this mutant in RGCs (Nagabhushana et al. 2010; Park et al. 2010). Transport of neurotrophins in the axons is crucial for the survival of neuronal cells. Blockade of axonal transport has been reported in glaucoma in humans and in experimental animal models (Pease et al. 2000). However, the molecules whose defective traffi cking by E50K optineurin causes RGC death in glaucoma need to be identifi ed. Is the defective traffi cking of neurotrophins, transferrin or their receptors (or some other associated molecules) responsible for RGC death? Another function of optineurin is regulation of signalling to the transcription factor NF-κB. NF-κB plays a key role in the expression of many genes involved in regulating immune response, apoptosis, cell cycle and its deregulation is involved in the pathogenesis of many diseases including some neurodegenerative disorders. Upon treatment of cells with TNFα, trimerization of TNF receptor results in the assembly of a signalling complex at the cytoplasmic side of the plasma membrane. In this complex RIP is ubiquitinated (by the addition of Lys63-linked ubiquitin chains) which then recruits NF-κB essential modulator (NEMO), the regulatory sub-unit of a kinase complex, IKK. This leads to activation of the catalytic subunits of the IKK complex, IKKα and β, that phosphorylate inhibitor of κB (IκB) resulting in its degradation. This enables nuclear translocation of NF-κB to activate transcription of target genes (Hayden and Ghosh 2008). Knockdown of optineurin increases basal as well as TNFα-induced NF-κB activity whereas overexpressed optineurin inhibits it. This negative regulation of NF-κB activity is believed to be the result of competition of optineurin with NEMO for binding to polyubiquitinated RIP (Zhu et al. 2007). C-terminal half of optineurin shows considerable homology with NEMO. However the regulation of NF-κB activity by optineurin is likely to be far more complex because optineurin interacts with two other negative regulators of NF-κB- CYLD, a deubiquitinase and A20, a ubiquitin editing enzyme (Chalasani et al. 2009). In human T-lymphotropic virus type 1 (HTLV-1) infected cells optineurin interacts with TAX1BP1 and a viral protein TAX1 resulting in sustained activation of NF-κB and ubiquitination of TAX1 (Journo et al. 2009). Thus role of optineurin in the regulation of NF-κB is cell type and stimulus dependent.

Immunoneutralization of insulin partially modifies dorsoventral patterning in developing frog embryos.

The non-transmembrane protein tyrosine phosphatase, PTP-S, is located predominantly in the cell nucleus in association with chromatin. Here we have analysed the expression of PTP-S upon mitogenic stimulation and during cell division cycle. During liver regeneration after partial hepatectomy, PTP–S mRNA levels increased 16-fold after 6 h (G1 phase) and declined thereafter. Upon stimulation of serum starved cells in culture with serum, PTP-S mRNA levels increased reaching a maximum during late G1 phase and declined thereafter. No significant change in PTP-S RNA levels was observed in growing cells during cell cycle. PTP–S protein levels were also found to increase upon mitogenic stimulation. Upon serum starvation for 72 h, PTP–S protein disappears from the nucleus and is seen in the cytoplasm; after 96 h of serum starvation the PTP-S protein disappears from the nucleus as well as cytoplasm. Refeeding of starved cells for 6 h results in reappearance of this protein in the nucleus. Our results suggest a role of this phosphatase during cell proliferation.

Nucleotide sequences of a cDNA coding for rat hck tyrosine kinase and characterization of its gene product.

The hck gene is member of src family of non-receptor type tyrosine kinases. Here we report the nucleotide sequence of the rat hck eDNA of 1.94 kb. The nuclcotide sequence shows an open reading frame coding for a polypeptide of 503 amino acids. A vector expressing a fusion protein of glutathione-S-transferase with 82 amino acids of the N-terminal region of hck (from amino acids 32 to 113) was constructed, Using this bacterially expressed fusion protein antibodies were prepared which recognize the cellular hck gene product. These antibodies identified, by immunoblotting, two polypeptides of 56 and 59 kDa in rat spleen where hck transcripts are present at high level. Immunoprecipitated hck polypeptides were enzymatically active and were autophosphorylated in the presence of ATP and Mg2+. 1mmunoprecipitated hck could phosphorylate exogenous substrates. Treatment of immunoprecipitated hck by a purified protein tyrosine phosphatase decreased its enzymatic acitivity. Our results suggest that the enzymatic activity of hck tyrosine kinase is regulated by phosphorylation and dephosphorylation.