Amelotin is expressed in retinal pigment epithelium and localizes to hydroxyapatite deposits in dry age-related macular degeneration

Rajapakse, Dinusha; Peterson, Katherine; Mishra, Sanghamitra; Fan, Jun‐Yu; Lerner, J; Campos, Maria M; Wistow, Graeme

doi:10.1016/j.trsl.2020.02.007

Cited by 8 publications

(9 citation statements)

References 51 publications

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“…binding of POS and subsequent phagocytosis through CD36 and MERTK [3,[6][7][8][9]. Our results also fit with gene expression profiles that have been published for ARPE19 cells [101], fetal and adult human RPE cells [102,103] as well as stem cell-derived RPE cells [104,105].…”

Section: Discussionsupporting

confidence: 89%

Immunochemical analysis of the integrin expression in retinal pigment epithelial cells

Heller

Heintz

Kwok

et al. 2020

Preprint

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Retinal pigment epithelial (RPE) cells have been used in disease modelling and transplantation studies in retinal diseases. Several types of RPE cells have been trialed, ranging from primary cells and immortalized cell lines to stem cell-derived RPE cells. During aging and in disease, the extracellular environment of the RPE cells changes, interfering with RPE cell adhesion. We hypothesize that this could be a key problem in transplantation studies that have reported lack of adhesion and survival of the transplanted RPE cells. Integrins are essential for the proper function of the RPE, mediating adhesion to Bruch's membrane, and the binding and subsequent phagocytosis of photoreceptor outer segments. Variability that has been found in clinical trials might be due to the variability of cell types used and their expression profiles of surface molecules. Here, we set out to analyze integrin expression in primary rat RPE cells and in the human cell line ARPE-19 using immunochemistry. We found that both cell types express integrins to varying degrees. After long-term culturing, ARPE-19 cells resemble mature RPE cells, and increase integrin expression. We hence argue that it is important to test the properties of these cells prior to transplantation to avoid failure of adhesion and to facilitate correct function.

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Section: Discussionsupporting

confidence: 89%

Immunochemical analysis of the integrin expression in retinal pigment epithelial cells

Heller

Heintz

Kwok

et al. 2020

Preprint

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“…Moreover, pathway analysis of genes that were significantly changed in either direction in the RNAseq dataset further confirm that low level GSK3 inhibition with CHIR results in substantial changes to the production and organization of the ECM as well as the basement membrane composition (Figure 5B). Additionally, genes of interest pertaining to sub-RPE deposits including amelotin (AMTN (49)) and fibronectin (FN (50)), ECM crosslinking enzymes such as lysyl oxidase (LOX (51)), and pro-EMT regulators such as WNT3 (52) and WNT5A (53) (Figure 5A) were all downregulated. Interestingly, in the upregulated gene set, two important genes for pigment formation in the RPE (54), tyrosinase (TYR) and TYR-related protein 1 (TYRP1), were significantly upregulated by CHIR treatment (Figure 5A).…”

Section: Resultsmentioning

confidence: 99%

GSK3 inhibition reduces ECM production and prevents age-related macular degeneration-like pathology

DiCesare,

Ortega,

Collier

et al. 2023

Preprint

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Malattia Leventinese/Doyne Honeycomb Retinal Dystrophy (ML/DHRD) is an age-related macular degeneration (AMD)-like retinal dystrophy caused by an autosomal dominant R345W mutation in the secreted glycoprotein, fibulin-3 (F3). To identify new small molecules that reduce F3 production from retinal pigmented epithelium (RPE) cells, we knocked-in a luminescent peptide tag (HiBiT) into the endogenous F3 locus which enabled simple, sensitive, and high throughput detection of the protein. The GSK3 inhibitor, CHIR99021 (CHIR), significantly reduced F3 burden (expression, secretion, and intracellular levels) in immortalized RPE and non-RPE cells. Low-level, long-term CHIR treatment promoted remodeling of the RPE extracellular matrix (ECM), reducing sub-RPE deposit-associated proteins (e.g., amelotin, complement component 3, collagen IV, and fibronectin), while increasing RPE differentiation factors (e.g., tyrosinase, and pigment epithelium derived factor). In vivo, treatment of 8 mo R345W+/+knockin mice with CHIR (25 mg/kg i.p., 1 mo) was well tolerated and significantly reduced R345W F3-associated AMD-like basal laminar deposit number and size, thereby preventing the main pathological feature in these mice. This is the first demonstration of small molecule-based prevention of AMD-like pathology in ML/DHRD mice and may herald a rejuvenation of interest in GSK3 inhibition for the treatment of neurodegenerative diseases, including, potentially AMD itself.

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“…We show that pressure-jump NMR spectroscopy provides detailed structural information on an oligomerizing species, amelotin (AMTN), a protein that plays a key role in the formation of enamel and hydroxyapatite mineralization. , Amelotin also has been implicated in the formation of hydroxyapatite deposits in age-related macular degeneration . While the protein is soluble and, at concentrations below ca.…”

Section: Resultsmentioning

confidence: 99%

Experimental NOE, Chemical Shift, and Proline Isomerization Data Provide Detailed Insights into Amelotin Oligomerization

et al. 2023

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Amelotin is an intrinsically disordered protein (IDP) rich in Pro residues and is involved in hydroxyapatite mineralization. It rapidly oligomerizes under physiological conditions of pH and pressure but reverts to its monomeric IDP state at elevated pressure. We identified a 105-residue segment of the protein that becomes ordered upon oligomerization, and we used pressure-jump NMR spectroscopy to measure long-range NOE contacts that exist exclusively in the oligomeric NMR-invisible state. The kinetics of oligomerization and dissociation were probed at the residue-specific level, revealing that the oligomerization process is initiated in the Cterminal half of the segment. Using pressure-jump NMR, the degree of order in the oligomer at the sites of Pro residues was probed by monitoring changes in cis/trans equilibria relative to the IDP state after long-term equilibration under oligomerizing conditions. Whereas most Pro residues revert to trans in the oligomeric state, Pro-49 favors a cis configuration and three Pro residues retain an unchanged cis fraction, pointing to their local lack of order in the oligomeric state. NOE contacts and secondary 13 C chemical shifts in the oligomeric state indicate the presence of an 11-residue α-helix, preceded by a small intramolecular antiparallel β-sheet, with slower formation of long-range intermolecular interactions to N-terminal residues. Although none of the models generated by AlphaFold2 for the amelotin monomer was consistent with experimental data, subunits of a hexamer generated by AlphaFold-Multimer satisfied intramolecular NOE and chemical shift data and may provide a starting point for developing atomic models for the oligomeric state.

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Amelotin is expressed in retinal pigment epithelium and localizes to hydroxyapatite deposits in dry age-related macular degeneration

Cited by 8 publications

References 51 publications

Immunochemical analysis of the integrin expression in retinal pigment epithelial cells

Immunochemical analysis of the integrin expression in retinal pigment epithelial cells

GSK3 inhibition reduces ECM production and prevents age-related macular degeneration-like pathology

Experimental NOE, Chemical Shift, and Proline Isomerization Data Provide Detailed Insights into Amelotin Oligomerization

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