Ординатура / Офтальмология / Английские материалы / Retinal Degeneration Disease_Hollyfield, Anderson, LaVail_1999

Figure 64.3. A working model for the transport of N-retinylidene-PE across disc membranes by ABCA4. N-retinylidene-PE binds to ABCA4 containing a nonexchangeable ADP in NBD1. ATP hydrolysis occurs in NBD2 resulting in a protein conformational change that initiates the flipping of N-retinylidene-PE across the disc membrane. Finally, N-retinylidene-PE and ADP dissociate returning ABCA4 its initial state.

4. SOME UNRESOLVED PROBLEMS

Although considerable progress has been made in understanding the role of ABCA4 in photoreceptor function and disease, several issues remain to be clarified. The direction of retinoid transport has not yet been determined experimentally. The current model favors the translocation of N-retinylidene-PE from the lumen to the cytoplasmic side of the disc membrane. This is based on the premise that N-retinylidene-PE trapped on the lumen side has to be transported or flipped to the cytoplasmic side of the disc membrane so that after dissociation, all-trans retinal can be reduced to all-trans retinol by retinol dehydrogenase and channeled into the visual cycle. Although this is a logical model, most eukaryotic ABC proteins transport substrates in the reverse direction i.e. from the cytoplasmic to the lumen or extracellular side of membranes. Hence, it is important to determine experimentally the direction and mechanism of retinoid transport by ABCA4. Additionally, it is important to know if ABCA4 interacts with other photoreceptor proteins and if so whether these proteins regulate the transport activity of ABCA4. The role of the various domains, including the two large extracellular domains upstream of the multispanning membrane domains and cytoplasmic segments downstream of the NBDs, on the structure, function and subcellular targeting of ABCA4 needs to be investigated. Finally, it is important to more fully understand how specific disease-causing mutations, affect the retinoid transport activity of ABCA4.

5. ACKNOWLEDGEMENTS

This work was supported by grants from the National Eye Institute (EY 02422), the Canadian Institutes for Health Research (MT 5822) and the Macular Vision Research Foundation.

6. REFERENCES

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Allikmets, R., Shroyer, N.F., Singh, N., Seddon, J.M., Lewis, R.A., Bernstein, P.S., Peiffer, A., Zabriskie, N.A., Li, Y., Hutchinson, A., Dean, M., Lupski, J.R. and Leppert, M. (1997a) Mutation of the Stargardt disease gene (ABCR) in age-related macular degeneration. Science, 277:805-1807.

Allikmets, R., Singh, N., Sun, H., Shroyer, N.F., Hutchinson, A., Chidambaram, A., Gerrard, B., Baird, L., Stauffer, D., Peiffer, A., Rattner, A., Smallwood, P., Li, Y., Anderson, K.L., Lewis, R.A., Nathans, J., Leppert, M., Dean, M. and Lupski, J.R. (1997b) A photoreceptor cell-specific ATP-binding transporter gene (ABCR) is mutated in recessive Stargardt macular dystrophy. Nat Genet, 15:236-246.

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Weng, J., Mata, N.L., Azarian, S.M., Tzekov, R.T., Birch, D.G. and Travis, G.H. (1999) Insights into the function of rim protein in photoreceptors and etiology of Stargardt’s Disease from the phenotype in abcr knockout mice. Cell, 98:3-23.

2. AIPL1 ASSOCIATES WITH THE CELL CYCLE REGULATOR NUB1

2.1. Expression of AIPL1 and NUB1 in the Human Retina

AIPL1 interacts with the NEDD8 ultimate buster protein 1 (NUB1) (Akey et al., 2002). NUB1 in turn interacts with the small ubiquitin-like protein NEDD8 and downregulates the targeted proteasomal degradation of NEDD8 and substrates that are conjugated to NEDD8 in a manner analogous to ubiquitination and sentrinization (Kamitani et al., 2001; Kito et al., 2001). All the known substrates for neddylation are members of the family of cullin (Cul) proteins, which are components of an SCF ubiquitin E3 ligase comprising Skp1, cullin, F-box protein, and ROC1 (Zheng et al., 2002). Specific substrates are targeted for SCFmediated ubiquitination and subsequent degradation depending on the identity of the cullin component. Ubiquitination of IkBa, b-catenin, cyclin D proteins, p27 (KIP1) and p21 (CIP1/WAF1) is catalysed by the Cul-1 SCF ubiquitin E3 ligase, the activity of which is dependent on NEDD8 conjugation of the cullin component. Ubiquitination and proteasomal degradation of hypoxia-inducible factor-1a (HIF1a) and cyclin E are catalysed by the Cul-2 and Cul-3 SCF complexes respectively. Hence, the conjugation of NEDD8 to cullins has been implicated in many important biological events including cell cycle regulation and cell signalling, and it has been suggested that the interaction of NUB1 with NEDD8 implicates NUB1 and AIPL1 in the regulation of these events.

The spatiotemporal distribution of NUB1 in the developing and adult human retina has been examined in parallel with that of AIPL1 (van der Spuy et al., 2002; van der Spuy et al., 2003). In the adult human retina, AIPL1 was localized specifically in the connecting cilia, inner segments, cell bodies, axons and spherules of the rod photoreceptors but could not be detected in the cone photoreceptors. However, LCA is characterised by a flat or severely attenuated scotopic and photopic electroretinogram (ERG) within the first year of life suggesting the severe and early degeneration or impaired function of the rod and cone photoreceptors. A spatiotemporal examination of AIPL1 expression in the developing human retina detected AIPL1 in a single layer of cells in the central retina at fetal week 11.8 corresponding to early presumptive cone photoreceptors, differentiation of which precedes that of rod photoreceptors. At this age, AIPL1 was not detected in mid-peripheral or peripheral regions of the presumptive retinal outer nuclear layer (ONL). As retinal development proceeded, the expression of AIPL1 in the developing photoreceptor ONL spread gradually from the central to peripheral retina, closely following the centroperipheral gradient of rod and cone photoreceptor differentiation. During development, AIPL1 was detected in both rod and cone photoreceptors, and co-localized with both short wavelength (S)-cone and long/medium wavelength (L/M)-cone opsin expression. These data suggested that while AIPL1 is important for normal rod and cone development, it is required for the maintenance of rod photoreceptors only in adults, and that a developmental switch in AIPL1 function occurs.

Unlike AIPL1, NUB1 was not photoreceptor-specific but was expressed ubiquitously in all tissues examined and in all the retinal cell types. NUB1 was detected predominantly in the nuclei of all cells. NUB1 was expressed in all retinal cell types during human retinal development and the spatiotemporal expression of NUB1 did not follow a centroperipheral gradient. Rather, NUB1 was detected equally from central to peripheral retina at each age examined. During development a potential gradient of high to low NUB1 expression was observed from the inner to outer retina that coincided with retinal cell differentiation. The

AIPL1 in the cytoplasm. The closely related AIPL1 homologue AIP was unable to interact with or modulate GFP-NUB1 nucleocytoplasmic distribution, suggesting that whilst the similarity between AIPL1 and AIP correlates with a conserved function in the modulation of nuclear translocation, the specificity for the client protein differs in each case.

In addition to its role in protein translocation, AIPL1 was also able to suppress inclusion formation by GFP-NUB1 N- and C-terminal fragments. GFP-NUB1-N (residues 1 – 306) encompassed two coiled-coil (cc) domains and the ubiquitin-like (UBL) domain in the N-terminus of GFP-NUB1, and in the absence of AIPL1 formed multiple, small inclusions that decorated the cytoplasmic side of the nucleus in a perinuclear fashion. GFP-NUB1-C (residues 347-601) encompassed two ubiquitin-associated (UBA) domains, an NLS and a PEST sequence towards the C-terminus of GFP-NUB1, and formed large, intranuclear inclusions in the absence of AIPL1. Increasing amounts of AIPL1 resulted in efficient suppression of GFP-NUB1-N and GFP-NUB1-C inclusion formation, and the redistribution and co-localisation of these fragments with AIPL1 in the cytoplasm. This effect was specific for the GFP-NUB1 fragments, as AIPL1 had no effect on the formation of inclusions by unrelated aggregation-prone proteins, GFP-Huntingtin-exon 1-Q103 and P23H mutant opsin-GFP. Whilst these aggregation-prone proteins were SDS insoluble, the GFP-NUB1 fragments were SDS soluble and recruited both ubiquitin and Hsp70, suggesting that they were targeted for proteasomal degradation. The AIPL1 homologue AIP had no effect on the formation of GFP-NUB1-N and -C inclusions.

Hence, AIPL1 was able to behave in a chaperone-like manner to modulate the nuclear translocation of GFP-NUB1 and suppress the formation of inclusions by GFP-NUB1 fragments. This function of AIPL1 was compromised by certain mutations. All of the AIPL1 disease-associated and engineered mutants generated were soluble and similar in their subcellular distribution to AIPL1, with the exception of the disease-associated mutation W278X. AIPL1(W278X) formed multiple, cytoplasmic inclusions that were SDS insoluble, suggesting that this mutant protein undergoes misfolding and aggregation and is nonfunctional. Two additional C-terminal truncation mutants not yet associated with disease, AIPL1(E317X) and AIPL1(Q329X) were also severely compromised, suggesting that C- terminal sequences in AIPL1 are necessary for the modulation of NUB1 nuclear localization and inclusion suppression. Some of the pathological mutations, including R302L and W278X, were less efficient that wild-type protein in modulating the subcellular distribution and nuclear translocation of NUB1 suggesting a possible mechanism for disease in these patients affecting NUB1-related events involving the regulation of cell signalling and cell growth. However, other pathological AIPL1 mutants including A197P, C239R, G262S and P351D12 were not defective in their ability to modulate the subcellular distribution of NUB1 or suppress the formation of inclusions by NUB1 fragments, suggesting that the basis for disease in these patients may involve an alternative protein interaction and related function for AIPL1.

3. AIPL1 CHAPERONE-LIKE FUNCTIONS IN POST-TRANSLATIONAL MODIFICATION AND POST-TRANSCRIPTIONAL REGULATION

It has been demonstrated that AIPL1 is specifically able to interact with and enhance the post-translational farnesylation of proteins in the retina (Ramamurthy et al., 2003). Protein prenylation facilitates protein-protein and protein-membrane interactions, and is

important in the maintenance of retinal cytoarchitecture and photoreceptor structure. It was shown that the ability of AIPL1 to interact with and enhance the processing of farnesylated proteins was severely compromised by certain pathogenic mutations including M79T and the non-functional W278X. It was suggested that AIPL1 may interact with the C-terminal prenylation motif in the cytosol dependent on the presence of a farnesyl transferase and either protect the farnesylated protein form proteasomal degradation in the cytosol, facilitate targeting of the protein to the ER for further processing, or chaperone the farnesylated protein to the target membrane (Figure 65.1). The AIPL1 mutants A197P and C239R, which were partially defective in their ability to interact with and facilitate the processing of farnesylated proteins, were functional with respect to their effect on NUB1. Another AIPL1 mutation, R302L, did not show any defect in protein farnesylation but was compromised in NUB1 function. Hence, the mechanisms of disease pathogenesis in patients with AIPL1 mutations may depend on the specific interacting partner and functional pathway affected.

Recently, mouse models of LCA with either complete or partial inactivation of AIPL1 expression have suggested that AIPL1 may also function as a potential chaperone for cGMP phosphodiesterase (PDE) (Figure 65.1) (Liu et al., 2004; Ramamurthy et al., 2004). In both models, normal retinal histology and morphological photoreceptor development were observed at birth, although no recordable photofunction could be detected in AIPL1-/- mice and the photoresponse onset and recovery was delayed in the rod photoreceptors of the AIPL1 hypomorphic mutant. Photoreceptor degeneration proceeded rapidly shortly after birth in the absence of AIPL1 but was significantly slowed in the presence of reduced levels of AIPL1. In both mouse models, all three subunits of the cGMP PDE holoenzyme (a, b and g) were reduced by a post-transcriptional mechanism before the onset of photoreceptor degeneration, suggesting that AIPL1 was necessary for the biosynthesis, assembly, or stabilization of PDE to proteasomal degradation. The PDE-a subunit is farnesylated and mutations that block farnesylation cause degradation of PDE-a protein in cultured cells (Qin and Baehr, 1994). However, LCA is more severe than retinitis pigmentosa (RP) caused by mutations in the PDE subunits.

4. CONCLUSIONS

In conclusion, it has been shown that AIPL1 is able to function in a chaperone-like manner to regulate the nuclear translocation of the cell cycle regulator NUB1, enhance the processing of farnesylated proteins and post-transcriptionally regulate the levels of PDE. The severity of disease in LCA patients with mutations in AIPL1 cannot be accounted for by each of these functions on their own, suggesting that multiple and complex AIPL1dependent mechanisms may be involved or that a single, as yet unidentified AIPL1dependent mechanism may underlie each of these chaperone-like functions. It is tempting to speculate that these processes could converge at the level of regulating PDE, for example the interaction of AIPL1 with farnesylated components of the PDE complex could be important for the stabilization of the PDE enzyme complex, similarly the interaction of AIPL1 with NUB1 could regulate a switch in the ubiquitin proteasome machinery and protection of PDE from degradation (Figure 65.1). Alternatively, AIPL1, like AIP and FKBP51/52, may act as a part of chaperone heterocomplex in several unrelated processes. Further experimentation is needed to define the critical roles of AIPL1 and their importance for the development and function of the retina.

476 J. VAN DER SPUY AND M.E. CHEETHAM

5. REFERENCES

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Sohocki, M.M., Bowne, SJ, Sullivan, L.S., Blackshaw, S., Cepko, C.L., Payne, A.M., Bhattacharya, S.S., Khaliq, S., Qasim Mehdi, S., Birch, D.G., Harrison, W.R., Elder, F.F., Heckenlively, J.R., and Daiger, P., 2000a, Mutations in a new photoreceptor-pineal gene on 17p cause Leber congenital amaurosis, Nat Genet. 24:79.

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east, Newfoundland, and India.9-13 CRALBP gene defects can tighten or abolish retinoid interactions.6

3. CRALBP LIGAND INTERACTIONS

3.1. Site Directed Mutagenesis

The lack of covalent interactions between the recombinant CRALBP (rCRALBP) and retinoid was demonstrated and residues Q210 and K221 associated with retinoid-binding by a combination of protein chemical modifications, site-directed mutagenesis, and UVvisible and fluorescence spectroscopy.14 Similar methods combined with heteronuclear single quantum correlation NMR and enzymatic assays with purified recombinant 11-cis- retinol dehydrogenase were used to demonstrate that rCRALBP residues W165, M208, M222, M225, and W244 influence retinoid-binding and substrate carrier function.15 Diseasecausing mutations R150Q and M225K abolish ligand-binding and R233W was found to significantly tighten rCRALBP retinoid affinity.6,9

3.2. Photo-Labeling of the CRALBP Ligand-Binding Pocket

Photo-labeling of rCRALBP with 3-diazo-4-keto-11-cis-retinal yielded covalent labeling of eight residues that were identified by mass spectrometry, namely Y179, F197, C198, M208, K221, M222, V223 and M225.16 Four of these photo-adducted residues were independently associated with ligand interactions as described above, supporting specific labeling of the ligand-binding cavity. An unexpected outcome was that each of the photoaffinity modified residues in rCRALBP exhibited one or more different adduct masses.16 We suspect that upon UV-irradiation, the resulting carbene radical moves freely throughout the conjugated polyene structure of the retinoid analogue and fragments at the time of attachment to the protein.

3.3. Structural Modeling of the CRALBP Retinoid-Binding Domain

The homology between CRALBP and other CRAL-TRIO family members spans about 185 amino acids and includes their respective ligand binding pockets.17 A model (Figure 66.1) of the CRALBP ligand binding domain16 was constructed based on crystal structures of homologues a-tocopherol transfer protein (aTTP), yeast Sec14, and supernatant protein factor. Five of the CRALBP residues associated with the retinoid binding pocket by biochemical analyses (W165, Y179, F197, M222, and M225) align directly with components identified in the ligand cavities of the CRAL-TRIO crystal structures16 and are very close to ligand in our model (average distance ~4.4 Å). Four other residues (C198, Q210, K221, and V223) align in relatively close proximity (average model distance from ligand ~7.6 Å). All nine residues line the ligand binding cavity in the model. Residues M208, R233W and W2446,15 are more distant from ligand in the structural model (average distance from ligand ~14.3 Å). M208 and W244 may be located at the entrance/exit to the ligand cavity and involved in conformational changes necessary for ligand binding and release.