Madhavi Latha Chalasani

Regulation of endocytic trafficking of transferrin receptor by optineurin and its impairment by a glaucoma-associated mutant

BackgroundOptineurin is a multifunctional protein involved in several functions such as vesicular trafficking from the Golgi to the plasma membrane, NF-κB regulation, signal transduction and gene expression. Mutations in optineurin are associated with glaucoma, a neurodegenerative eye disease that causes blindness. Genetic evidence suggests that the E50K (Glu50Lys) is a dominant disease-causing mutation of optineurin. However, functional alterations caused by mutations in optineurin are not known. Here, we have analyzed the role of optineurin in endocytic recycling and the effect of E50K mutant on this process.ResultsWe show that the knockdown of optineurin impairs trafficking of transferrin receptor to the juxtanuclear region. A point mutation (D474N) in the ubiquitin-binding domain abrogates localization of optineurin to the recycling endosomes and interaction with transferrin receptor. The function of ubiquitin-binding domain of optineurin is also needed for trafficking of transferrin to the juxtanuclear region. A disease causing mutation, E50K, impairs endocytic recycling of transferrin receptor as shown by enlarged recycling endosomes, slower dynamics of E50K vesicles and decreased transferrin uptake by the E50K-expressing cells. This impaired trafficking by the E50K mutant requires the function of its ubiquitin-binding domain. Compared to wild type optineurin, the E50K optineurin shows enhanced interaction and colocalization with transferrin receptor and Rab8. The velocity of Rab8 vesicles is reduced by co-expression of the E50K mutant. These results suggest that the E50K mutant affects Rab8-mediated transferrin receptor trafficking.ConclusionsOur results suggest that optineurin regulates endocytic trafficking of transferrin receptor to the juxtanuclear region. The E50K mutant impairs trafficking at the recycling endosomes due to altered interactions with Rab8 and transferrin receptor. These results also have implications for the pathogenesis of glaucoma caused by the E50K mutation because endocytic recycling is vital for maintaining homeostasis.

Optineurin and Its Mutants: Molecules Associated with Some Forms of Glaucoma

Aims: Optineurin is a gene (OPTN) associated with normal-tension glaucoma and primary open-angle glaucoma. The aim of our study is to understand the functions of the protein optineurin in retinal ganglion cells, and the modifications that come about in its functions when mutations occur in its sequence. Methods: We have worked with the rat retinal ganglion cell line RGC5, as well as with some others such as HeLa (human cancer cell line), COS-1 (monkey kidney cell line), IMR-32 (human neuroblastoma cell line), and D407 (human retinal pigment epithelial cell line). The cDNAs of wild-type OPTN, and of its mutants H26D, E50K, R454Q and H486R, were transfected into each of these cell lines and the effects studied. Localization of the proteins in cells was monitored by confocal microscopy and cell imaging, and their interaction with other cellular proteins by yeast two-hybrid assay and co-immunoprecipitation. Results: We found optineurin to interact with several proteins, which are involved in NF-κB regulation, vesicular trafficking at recycling endosomes, immune response and transcription regulation. Some of these proteins showed altered/reduced interactions with E50K and H486R mutants. E50K, a severe phenotype-related mutant of optineurin, was found to selectively kill RGC5 cells through oxytosis and apoptosis, while none of the other mutants did so. Conclusions: Our results indicate that several independent mechanisms are likely to be involved in the pathogenesis of glaucoma caused by mutations in optineurin.

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

Summary 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).

E50K-OPTN-Induced Retinal Cell Death Involves the Rab GTPase-Activating Protein, TBC1D17 Mediated Block in Autophagy

The protein optineurin coded by OPTN gene is involved in several functions including regulation of endocytic trafficking, autophagy and signal transduction. Certain missense mutations in the gene OPTN cause normal tension glaucoma. A glaucoma-causing mutant of optineurin, E50K, induces death selectively in retinal cells. This mutant induces defective endocytic recycling of transferrin receptor by causing inactivation of Rab8 mediated by the GTPase-activating protein, TBC1D17. Here, we have explored the mechanism of E50K-induced cell death. E50K-OPTN-induced cell death was inhibited by co-expression of a catalytically inactive mutant of TBC1D17 and also by shRNA mediated knockdown of TBC1D17. Endogenous TBC1D17 colocalized with E50K-OPTN in vesicular structures. Co-expression of transferrin receptor partially protected against E50K-induced cell death. Overexpression of the E50K-OPTN but not WT-OPTN inhibited autophagy flux. Treatment of cells with rapamycin, an inducer of autophagy, reduced E50K-OPTN-induced cell death. An LC3-binding-defective mutant of E50K-OPTN showed reduced cell death, further suggesting the involvement of autophagy. TBC1D17 localized to autophagosomes and inhibited autophagy flux dependent on its catalytic activity. Knockdown of TBC1D17 rescued cells from E50K-mediated inhibition of autophagy flux. Overall, our results suggest that E50K mutant induced death of retinal cells involves impaired autophagy as well as impaired transferrin receptor function. TBC1D17, a GTPase-activating protein for Rab GTPases, plays a crucial role in E50K-induced impaired autophagy and cell death.

A cataract-causing connexin 50 mutant is mislocalized to the ER due to loss of the fourth transmembrane domain and cytoplasmic domain.

Mutations in the eye lens gap junction protein connexin 50 cause cataract. Earlier we identified a frameshift mutant of connexin 50 (c.670insA; p.Thr203AsnfsX47) in a family with autosomal recessive cataract. The mutant protein is smaller and contains 46 aberrant amino acids at the C‐terminus after amino acid 202. Here, we have analysed this frameshift mutant and observed that it localized to the endoplasmic reticulum (ER) but not in the plasma membrane. Moreover, overexpression of the mutant resulted in disintegration of the ER‐Golgi intermediate compartment (ERGIC), reduction in the level of ERGIC‐53 protein and breakdown of the Golgi in many cells. Overexpression of the frameshift mutant partially inhibited the transport of wild type connexin 50 to the plasma membrane. A deletion mutant lacking the aberrant sequence showed predominant localization in the ER and inhibited anterograde protein transport suggesting, therefore, that the aberrant sequence is not responsible for improper localization of the frameshift mutant. Further deletion analysis showed that the fourth transmembrane domain and a membrane proximal region (231–294 amino acids) of the cytoplasmic domain are needed for transport from the ER and localization to the plasma membrane. Our results show that a frameshift mutant of connexin 50 mislocalizes to the ER and causes disintegration of the ERGIC and Golgi. We have also identified a sequence of connexin 50 crucial for transport from the ER and localization to the plasma membrane.

Contents Vol. 42, 2009

Anatomy, Pathology and Cell Biology A. Prescott, Dundee Biochemistry, Molecular Biology and Molecular Genetics J. Graw, Neuherberg Clinical and Epidemiological Research M. Kojima, Kahoku Clinical Retina P. Wiedemann, Leipzig Cornea and Ocular Surface C. Marfurt, Gary, Ind. Glaucoma M. Coroneo, Sydney Immunology and Microbiology U. Pleyer, Berlin Lens and Cataract S. Varma, Baltimore, Md. Miscellaneous U. Pleyer, Berlin Neuro-Ophthalmology and Vision Sciences P. Aydin, Ankara Ocular Oncology M. Jager, Leiden Physiology, Pharmacology and Toxicology A. Wegener, Bonn Retina and Retinal Cell Biology M. Boulton, Gainesville, Fla. P. Wiedemann, Leipzig Editorial Board