BiP prevents rod opsin aggregation

Mutations in rod opsin—the light-sensitive protein of rod cells—cause retinitis pigmentosa. Many rod opsin mutations lead to protein misfolding, and therefore it is impor¬tant to understand the role of molecular chaperones in rod opsin biogenesis. We show that BiP (HSPA5) prevents the aggregation of rod opsin. Cleavage of BiP with the subtilase cyto¬toxin SubAB results in endoplasmic reticulum (ER) retention and ubiquitylation of wild-type (WT) rod opsin (WT–green fluorescent protein [GFP]) at the ER. Fluorescence recovery after photobleaching reveals that WT-GFP is usually mobile in the ER. By contrast, depletion of BiP activity by treatment with SubAB or coexpression of a BiP ATPase mutant, BiP(T37G), de¬creases WT-GFP mobility to below that of the misfolding P23H mutant of rod opsin (P23H-GFP), which is retained in the ER and can form cytoplasmic ubiquitylated inclusions. SubAB treatment of P23H-GFP–expressing cells decreases the mobility of the mutant protein further and leads to ubiquitylation throughout the ER. Of interest, BiP overexpression increases the mobility of P23H-GFP, suggesting that it can reduce mutant rod opsin aggregation. Therefore inhibition of BiP function results in aggregation of rod opsin in the ER, which suggests that BiP is important for maintaining the solubility of rod opsin in the ER.Dimitra Athanasiou, Maria Kosmaoglou, Naheed Kanuga, Sergey S. Novoselov, Adrienne W. Paton, James C. Paton, J. Paul Chapple and Michael E. Cheetha

Inactivation of VCP/ter94 Suppresses Retinal Pathology Caused by Misfolded Rhodopsin in Drosophila

The most common Rhodopsin (Rh) mutation associated with autosomal dominant retinitis pigmentosa (ADRP) in North America is the substitution of proline 23 by histidine (RhP23H). Unlike the wild-type Rh, mutant RhP23H exhibits folding defects and forms intracellular aggregates. The mechanisms responsible for the recognition and clearance of misfolded RhP23H and their relevance to photoreceptor neuron (PN) degeneration are poorly understood. Folding-deficient membrane proteins are subjected to Endoplasmic Reticulum (ER) quality control, and we have recently shown that RhP23H is a substrate of the ER–associated degradation (ERAD) effector VCP/ter94, a chaperone that extracts misfolded proteins from the ER (a process called retrotranslocation) and facilitates their proteasomal degradation. Here, we used Drosophila, in which Rh1P37H (the equivalent of mammalian RhP23H) is expressed in PNs, and found that the endogenous Rh1 is required for Rh1P37H toxicity. Genetic inactivation of VCP increased the levels of misfolded Rh1P37H and further activated the Ire1/Xbp1 ER stress pathway in the Rh1P37H retina. Despite this, Rh1P37H flies with decreased VCP function displayed a potent suppression of retinal degeneration and blindness, indicating that VCP activity promotes neurodegeneration in the Rh1P37H retina. Pharmacological treatment of Rh1P37H flies with the VCP/ERAD inhibitor Eeyarestatin I or with the proteasome inhibitor MG132 also led to a strong suppression of retinal degeneration. Collectively, our findings raise the possibility that excessive retrotranslocation and/or degradation of visual pigment is a primary cause of PN degeneration

Using molecular chaperones to manipulate rhodopsin retinitis pigmentosa

The experiments described in this thesis were designed in order to test the hypothesis that molecular chaperones are involved in the biogenesis of rhodopsin and may be used in the treatment of rhodopsin retinitis pigmentosa. Rhodopsin is the prototypical G-protein coupled receptor found at high concentration in the outer segments of rod photoreceptor cells. Rhodopsin is made up of the rod opsin apoprotein and 11-cis-retinal, the photoactive ligand. Rhodopsin initiates the phototransduction cascade under dim light conditions and mutations in its primary sequence have been linked to the neurodegenerative blinding disease, retinitis pigmentosa. Mutations such as P23H, cause the misfolding of the protein, resulting in its retention in the endoplasmic reticulum of heterologous expression systems and the inner segment of photoreceptor cells. Whilst selecting suitable modifiers, the subcellular compartments occupied by rhodopsin during its biogenesis and the chaperones resident in these, were considered. Calnexin is a central component of the quality control machinery in the endoplasmic reticulum. As calnexin has been widely documented to assist in the maturation of nascent glycoproteins, mouse embryonic fibroblast cells were used, which expressed a truncated version of calnexin, unable to bind client glycoproteins. The expression of rod opsin was compared in cells expressing truncated calnexin and in their wild-type counterparts, assessing the contribution of calnexin in the subcellular localization and biochemical profile of rod opsin. Calnexin was found to be dispensable for the maturation and folding of rod opsin. EDEM1, the ER-degradation enhancing mannosidase α-like 1 protein, has been shown to accelerate the degradation of misfolded glycoproteins, extracting these from futile folding attempts in the calnexin cycle. EDEM1 was found to enhance the degradation of P23H rod opsin and importantly, promoted the cell surface expression of any remaining P23H molecules which escaped degradation. The localization of EDEM1 in murine retina was determined to be within a subset of the inner segment and rhodopsin was found to form a physiological immune complex in porcine retina. The binding protein, BiP, associates with nascent proteins as these are translated and translocated in the ER lumen. A toxin that efficiently cleaves BiP in two fragments was used in order to probe the effects of BiP deletion on the biogenesis of wild-type and mutant rod opsin. Wild-type rod opsin was found retained in the endoplasmic reticulum in the absence of functional BiP and was misfolded as ubiquitin was recruited to the endoplasmic reticulum surface from a previous diffuse localization. Therefore BiP appears to be critical for maintaining rod opsin in a folding competent state. A chaperone on the cytoplasmic face of the endoplasmic reticulum, namely HSJ1b, has previously been shown to result in the stalling of wild-type and mutant rod opsin folding. We have investigated the effects of coexpressing CHIP, the carboxy-terminus of Hsp70-interacting protein. CHIP is an E3 ligase, which has been shown to present misfolded proteins to the proteasome for degradation, via an association with the Hsp70 machinery and HSJ1b initiates the process by stimulating ATP hydrolysis by Hsp70. In the presence of HSJ1b, expression of CHIP resulted in the degradation of rod opsin by the proteasome. Hence chaperones in the endoplasmic reticulum lumen and the cytoplasm can be used to manipulate mutant P23H rod opsin and may be used in the treatment of rhodopsin RP

Carcinosarcoma of the bladder

We present a case of carcinosarcoma of the bladder, the first in our experience. This tumour is generally considered to be a rare one, has uncertain aetiology and poor prognosis. At present, radical cystectomy is the treatment of choice. © 1995 Akadémiai Kiadó

Differential expression of two distinct functional isoforms of melanopsin (Opn4) in the mammalian retina.

Melanopsin is the photopigment that confers photosensitivity to a subset of retinal ganglion cells (pRGCs) that regulate many non-image-forming tasks such as the detection of light for circadian entrainment. Recent studies have begun to subdivide the pRGCs on the basis of morphology and function, but the origin of these differences is not yet fully understood. Here we report the identification of two isoforms of melanopsin from the mouse Opn4 locus, a previously described long isoform (Opn4L) and a novel short isoform (Opn4S) that more closely resembles the sequence and structure of rat and human melanopsins. Both isoforms, Opn4L and Opn4S, are expressed in the ganglion cell layer of the retina, traffic to the plasma membrane and form a functional photopigment in vitro. Quantitative PCR revealed that Opn4S is 40 times more abundant than Opn4L. The two variants encode predicted proteins of 521 and 466 aa and only differ in the length of their C-terminal tails. Antibodies raised to isoform-specific epitopes identified two discrete populations of melanopsin-expressing RGCs, those that coexpress Opn4L and Opn4S and those that express Opn4L only. Recent evidence suggests that pRGCs show a range of anatomical subtypes, which may reflect the functional diversity reported for mouse Opn4-mediated light responses. The distinct isoforms of Opn4 described in this study provide a potential molecular basis for generating this diversity, and it seems likely that their differential expression plays a role in generating the variety of pRGC light responses found in the mammalian retina

Calnexin Improves the Folding Efficiency of Mutant Rhodopsin in the Presence of Pharmacological Chaperone 11-cis-Retinal*

The lectin chaperone calnexin (Cnx) is important for quality control of glycoproteins, and the chances of correct folding of a protein increase the longer the protein interacts with Cnx. Mutations in glycoproteins increase their association with Cnx, and these mutant proteins are retained in the endoplasmic reticulum. However, until now, the increased interaction with Cnx was not known to increase the folding of mutant glycoproteins. Because many human diseases result from glycoprotein misfolding, a Cnx-assisted folding of mutant glycoproteins could be beneficial. Mutations of rhodopsin, the glycoprotein pigment of rod photoreceptors, cause misfolding resulting in retinitis pigmentosa. Despite the critical role of Cnx in glycoprotein folding, surprisingly little is known about its interaction with rhodopsin or whether this interaction could be modulated to increase the folding of mutant rhodopsin. Here, we demonstrate that Cnx preferentially associates with misfolded mutant opsins associated with retinitis pigmentosa. Furthermore, the overexpression of Cnx leads to an increased accumulation of misfolded P23H opsin but not the correctly folded protein. Finally, we demonstrate that increased levels of Cnx in the presence of the pharmacological chaperone 11-cis-retinal increase the folding efficiency and result in an increase in correct folding of mutant rhodopsin. These results demonstrate that misfolded rather than correctly folded rhodopsin is a substrate for Cnx and that the interaction between Cnx and mutant, misfolded rhodopsin, can be targeted to increase the yield of folded mutant protein