RESUMEN
Fibulin-3 (FBLN3), also known as EFEMP1, is a secreted extracellular matrix (ECM) glycoprotein that contains forty cysteine residues. These cysteines, which are distributed across one atypical and five canonical calcium-binding epidermal growth factor (EGF) domains, are important for regulating FBLN3 structure, secretion, and presumably function. As evidence of this importance, a rare homozygous p.C55R mutation in FBLN3 negates its function, alters disulfide bonding, and causes marfanoid syndrome. Additional studies suggest that heterozygous premature stop codon mutations in FBLN3 may also cause similar, albeit less severe, connective tissue disorders. Interestingly, a series of twenty-four cysteine mutations in FBLN3 have been identified in the human population and published in the Clinical Variation (ClinVar) and gnomAD databases. We tested how seven of these cysteine mutants (five loss-of-cysteine variants: C42Y, C190R, C218R, C252F, and C365S, two gain-of-cysteine variants: R358C, Y369C) and two newly developed mutations (G57C and Y397C) altered FBLN3 secretion, disulfide bonding, MMP2 zymography, and stress response activation Surprisingly, we found a wide variety of biochemical behaviors: i) loss-of-cysteine variants correlated with an increased likelihood of disulfide dimer formation, ii) N-terminal mutations were less likely to disrupt secretion, and were less prone to aggregation, iii) in contrast to wild-type FBLN3, multiple, but not all variants failed to induce MMP2 levels in cell culture, and iv) C-terminal mutations (either loss or gain of cysteines) were more prone to significant secretion defects, intracellular accumulation/misfolding, and stress response activation. These results provide molecular and biochemical insight into FBLN3 folding, secretion, and function for many cysteine mutations found in the human population, some of which may increase the likelihood of subclinical connective tissue or other FBLN3-associated haploinsufficiency diseases.
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Malattia Leventinese/Doyne honeycomb retinal dystrophy (ML/DHRD) is an age-related macular degeneration-like (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 in retinal pigmented epithelium (RPE) cells, we knocked-in a luminescent peptide tag (HiBiT) into the endogenous F3 locus that 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, 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-month-old 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 an important 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 retinal degenerative diseases, including potentially AMD itself.
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Proteínas de la Matriz Extracelular , Matriz Extracelular , Degeneración Macular , Epitelio Pigmentado de la Retina , Animales , Ratones , Epitelio Pigmentado de la Retina/patología , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/efectos de los fármacos , Degeneración Macular/patología , Degeneración Macular/genética , Degeneración Macular/tratamiento farmacológico , Degeneración Macular/metabolismo , Humanos , Proteínas de la Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Matriz Extracelular/efectos de los fármacos , Piridinas/farmacología , Pirimidinas/farmacología , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/genética , Modelos Animales de Enfermedad , Distrofias Retinianas/metabolismo , Distrofias Retinianas/patología , Distrofias Retinianas/genética , Drusas del Disco Óptico/congénitoRESUMEN
Vimentin has been reported to play diverse roles in cell processes such as spreading, migration, cell-matrix adhesion, and fibrotic transformation. Here, we assess how vimentin impacts cell spreading, morphology, and myofibroblast transformation of human corneal fibroblasts. Overall, although knockout (KO) of vimentin did not dramatically impact corneal fibroblast spreading and mechanical activity (traction force), cell elongation in response to PDGF was reduced in vimentin KO cells as compared to controls. Blocking vimentin polymerization using Withaferin had even more pronounced effects on cell spreading and also inhibited cell-induced matrix contraction. Furthermore, although absence of vimentin did not completely block TGFß-induced myofibroblast transformation, the degree of transformation and amount of αSMA protein expression was reduced. Proteomics showed that vimentin KO cells cultured in TGFß had a similar pattern of protein expression as controls. One exception included periostin, an ECM protein associated with wound healing and fibrosis in other cell types, which was highly expressed only in Vim KO cells. We also demonstrate for the first time that LRRC15, a protein previously associated with myofibroblast transformation of cancer-associated fibroblasts, is also expressed by corneal myofibroblasts. Interestingly, proteins associated with LRRC15 in other cell types, such as collagen, fibronectin, ß1 integrin and α11 integrin, were also upregulated. Overall, our data show that vimentin impacts both corneal fibroblast spreading and myofibroblast transformation. We also identified novel proteins that may regulate corneal myofibroblast transformation in the presence and/or absence of vimentin.
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Córnea , Fibroblastos , Miofibroblastos , Vimentina , Humanos , Vimentina/metabolismo , Miofibroblastos/metabolismo , Miofibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/efectos de los fármacos , Córnea/citología , Córnea/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Movimiento Celular/efectos de los fármacos , Witanólidos/farmacología , Células CultivadasRESUMEN
Fibulin-3 (FBLN3, aka EFEMP1) is a secreted extracellular matrix (ECM) glycoprotein implicated in ocular diseases including glaucoma and age-related macular degeneration. Yet surprisingly, little is known about its native biology, expression patterns, and localization in the eye. To overcome these shortcomings, we conducted gene expression analysis and immunohistochemistry for FBLN3 in ocular tissues from mice, pigs, non-human primates, and humans. Moreover, we evaluated age-related changes in FBLN3 and FBLN3-related ECM remodeling enzymes/inhibitors in aging mice. We found that FBLN3 displayed distinct staining patterns consistent across the mouse retina, particularly in the ganglion cell layer and inner nuclear layer (INL). In contrast, human retinas exhibited a unique staining pattern, with enrichment of FBLN3 in the retinal pigment epithelium (RPE), INL, and outer nuclear layer (ONL) in the peripheral retina. This staining transitioned to the outer plexiform layer (OPL) in the central retina/macula, and was accompanied by reduced RPE immunoreactivity approaching the fovea. Surprisingly, we found significant age-related increases in FBLN3 expression and protein abundance in the mouse retina which was paralleled by reduced transcript levels of FBLN3-degrading enzymes (i.e., Mmp2 and Htra1). Our findings highlight important species-dependent, retinal region-specific, and age-related expression and localization patterns of FBLN3 which favor its accumulation during aging. These findings contribute to a better understanding of FBLN3's role in ocular pathology and provide valuable insights for future FBLN3 research.
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Envejecimiento , Proteínas de la Matriz Extracelular , Animales , Humanos , Ratones , Proteínas de la Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/genética , Envejecimiento/metabolismo , Envejecimiento/genética , Retina/metabolismo , Porcinos , Masculino , Ratones Endogámicos C57BL , Degeneración Macular/metabolismo , Degeneración Macular/patología , Degeneración Macular/genética , Epitelio Pigmentado de la Retina/metabolismo , Femenino , Especificidad de la Especie , AncianoRESUMEN
We present a method of in vitro/in vivo protein detection by pairing CRISPR-Cas9 genome editing with the NanoBiT system. We describe steps for cell culturing, in vitro CRISPR-Cas9 ribonucleoprotein delivery, cell monitoring, efficiency assessments, and edit analysis through HiBiT assays. We then detail procedures to determine edit specificity through genomic DNA analysis, small interfering RNA reverse transfection, and HiBiT blotting. This protocol is simple to execute and multifunctional, and it enables high-throughput screens on endogenous proteins to be conducted with ease.
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Sistemas CRISPR-Cas , Edición Génica , Sistemas CRISPR-Cas/genética , Edición Génica/métodos , Humanos , Proteínas/genética , Proteínas/metabolismo , Células HEK293 , Transfección/métodosRESUMEN
Activated microglia have been implicated in the pathogenesis of age-related macular degeneration (AMD), diabetic retinopathy, and other neurodegenerative and neuroinflammatory disorders, but our understanding of the mechanisms behind their activation is in infant stages. With the goal of identifying novel genes associated with microglial activation in the retina, we applied a semiquantitative fundus spot scoring scale to an unbiased, state-of-the-science mouse forward genetics pipeline. A mutation in the gene encoding the E3 ubiquitin ligase Herc3 led to prominent accumulation of fundus spots. CRISPR mutagenesis was used to generate Herc3-/- mice, which developed prominent accumulation of fundus spots and corresponding activated Iba1 + /CD16 + subretinal microglia, retinal thinning on OCT and histology, and functional deficits by Optomotory and electrophysiology. Bulk RNA sequencing identified activation of inflammatory pathways and differentially expressed genes involved in the modulation of microglial activation. Thus, despite the known expression of multiple E3 ubiquitin ligases in the retina, we identified a non-redundant role for Herc3 in retinal homeostasis. Our findings are significant given that a dysregulated ubiquitin-proteasome system (UPS) is important in prevalent retinal diseases, in which activated microglia appear to play a role. This association between Herc3 deficiency, retinal microglial activation and retinal degeneration merits further study.
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Microglía , Degeneración Retiniana , Animales , Humanos , Ratones , Microglía/metabolismo , Retina/patología , Degeneración Retiniana/patología , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinas/metabolismoRESUMEN
The formation of higher-order protein assemblies (commonly called protein aggregates) has long been associated with disease states, particularly in neurodegenerative disorders. Within the eye, protein aggregation has also been implicated in various retinal degenerative diseases ranging from retinitis pigmentosa (RP) to Malattia Leventinese/Doyne Honeycomb Retinal Dystrophy (ML/DHRD) to age-related macular degeneration (AMD). Yet, many essential cellular processes including transcription, translation, and the formation of non-membrane bound organelles require the formation of functional, non-pathologic protein aggregates to maintain cellular homeostasis. Thus, functional protein aggregates, also called condensates, likely play essential roles in maintaining normal retina function. However, currently, there is a critical gap in our knowledge: What proteins form higher-order assemblies under normal conditions within the retina and what function do these structures serve? Herein, we present data suggesting that protein aggregation is identifiable in multiple retinal layers of normal, healthy murine retina, and briefly discuss the potential contributions of aggregated proteins to normal retinal function, with a focus on the photoreceptor inner and outer segment.
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Degeneración Macular , Degeneración Retiniana , Humanos , Ratones , Animales , Degeneración Retiniana/genética , Degeneración Retiniana/patología , Agregado de Proteínas , Degeneración Macular/genética , Degeneración Macular/patología , Retina/patología , Proteínas AmiloidogénicasRESUMEN
Microglia play a role in the pathogenesis of many retinal diseases. Fundus spots in mice often correlate with the accumulation of activated subretinal microglia. Here we use a semiquantitative fundus spot scoring scale in combination with an unbiased, state-of-the-science forward genetics pipeline to identify causative associations between chemically induced mutations and fundus spot phenotypes. Among several associations, we focus on a missense mutation in Lipe linked to an increase in yellow fundus spots in C57BL/6J mice. Lipe-/- mice generated using CRISPR-Cas9 technology are found to develop accumulation of subretinal microglia, a retinal degeneration with decreased visual function, and an abnormal retinal lipid profile. We establish an indispensable role of Lipe in retinal/RPE lipid homeostasis and retinal health. Further studies using this new model will be aimed at determining how lipid dysregulation results in the activation of subretinal microglia and whether these microglia also play a role in the subsequent retinal degeneration.
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Degeneración Retiniana , Animales , Ratones , Modelos Animales de Enfermedad , Pruebas Genéticas , Lípidos , Ratones Endogámicos C57BL , Degeneración Retiniana/genética , Degeneración Retiniana/patologíaRESUMEN
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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The standardization of variant curation criteria is essential for accurate interpretation of genetic results and clinical care of patients. The variant curation guidelines developed by the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) in 2015 are widely used but are not gene specific. To address this issue, the Clinical Genome Resource (ClinGen) Variant Curation Expert Panels (VCEP) have been tasked with developing gene-specific variant curation guidelines. The Glaucoma VCEP was created to develop rule specifications for genes associated with primary glaucoma, including myocilin (MYOC), the most common cause of Mendelian glaucoma. Of the 28 ACMG/AMP criteria, the Glaucoma VCEP adapted 15 rules to MYOC and determined 13 rules not applicable. Key specifications included determining minor allele frequency thresholds, developing an approach to counting probands and segregations, and reviewing functional assays. The rules were piloted on 81 variants and led to a change in classification in 40% of those that were classified in ClinVar, with functional evidence influencing the classification of 18 variants. The standardized variant curation guidelines for MYOC provide a framework for the consistent application of the rules between laboratories, to improve MYOC genetic testing in the management of glaucoma.
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Genoma Humano , Glaucoma , Humanos , Pruebas Genéticas/métodos , Variación Genética , Glaucoma/diagnóstico , Glaucoma/genética , Patología Molecular , Estados UnidosRESUMEN
Destabilizing domains (DDs) are an attractive strategy allowing for positive post-transcriptional small molecule-regulatable control of a fusion protein's abundance. However, in many instances, the currently available DDs suffer from higher-than-desirable basal levels of the fusion protein. Accordingly, we redesigned the E. coli dihydrofolate reductase (ecDHFR) DD by introducing a library of â¼1200 random ecDHFR mutants fused to YFP into CHO cells. Following successive rounds of fluorescence-activated cell sorting, we identified six new ecDHFR DD clones with significantly enhanced proteasomal turnover in the absence of a stabilizing ligand, trimethoprim (TMP). One of these clones, designated as "C12", contained four unique missense mutations (W74R/T113S/E120D/Q146L) and demonstrated a significant 2.9-fold reduction in basal levels compared to the conventional ecDHFR DD (i.e., R12Y/G67S/Y100I). This domain was similarly responsive to TMP with respect to dose response and maximal stabilization, indicating an overall enhanced dynamic range. Interestingly, both computational and wet-lab experiments identified the W74R and T113S mutations of C12 as the main contributors toward its basal destabilization. However, the combination of all the C12 mutations was required to maintain both its enhanced degradation and TMP stabilization. We further demonstrate the utility of C12 by fusing it to IκBα and Nrf2, two stress-responsive proteins that have previously been challenging to regulate. In both instances, C12 significantly enhanced the basal turnover of these proteins and improved the dynamic range of regulation post stabilizer addition. These advantageous features of the C12 ecDHFR DD variant highlight its potential for replacing the conventional N-terminal ecDHFR DD and improving the use of DDs overall, not only as a chemical biology tool but for gene therapy avenues as well.
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Escherichia coli , Tetrahidrofolato Deshidrogenasa , Animales , Cricetinae , Tetrahidrofolato Deshidrogenasa/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Inhibidor NF-kappaB alfa , Factor 2 Relacionado con NF-E2 , Ligandos , Cricetulus , Trimetoprim/farmacologíaRESUMEN
Fibulin-3 (F3 or EFEMP1) is a disulfide-rich, secreted glycoprotein necessary for maintaining extracellular matrix (ECM) and connective tissue integrity. Three studies have identified distinct autosomal recessive F3 mutations in individuals with Marfan Syndrome-like phenotypes. Herein, we characterize how one of these mutations, c.163T>C; p.Cys55Arg (C55R), disrupts F3 secretion, quaternary structure, and function by forming unique extracellular disulfide-linked homodimers. Dual cysteine mutants suggest that the C55R-induced disulfide species forms because of the new availability of Cys70 on adjacent F3 monomers. Surprisingly, mutation of single cysteines located near Cys55 (i.e., Cys29, Cys42, Cys48, Cys61, Cys70, Cys159, and Cys171) also produced similar extracellular disulfide-linked dimers, suggesting that this is not a phenomenon isolated to the C55R mutant. To assess C55R functionality, F3 knockout (KO) retinal pigmented epithelial (RPE) cells were generated, followed by reintroduction of wild-type (WT) or C55R F3. F3 KO cells produced lower levels of the ECM remodeling enzyme, matrix metalloproteinase 2, and reduced formation of collagen VI ECM filaments, both of which were partially rescued by WT F3 overexpression. However, C55R F3 was unable to compensate for these same ECM-related defects. Our results highlight the unique behavior of particular cysteine mutations in F3 and uncover potential routes to restore C55R F3 loss-of-function.
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Cisteína , Disulfuros , Humanos , Cisteína/genética , Metaloproteinasa 2 de la Matriz/genética , Proteínas de la Matriz Extracelular/genética , Matriz Extracelular/genética , MutaciónRESUMEN
The Escherichia coli dihydrofolate reductase (DHFR) destabilizing domain (DD) serves as a promising approach to conditionally regulate protein abundance in a variety of tissues. To test whether this approach could be effectively applied to a wide variety of aged and disease-related ocular mouse models, we evaluated the DHFR DD system in the eyes of aged mice (up to 24 months), a light-induced retinal degeneration (LIRD) model, and two genetic models of retinal degeneration (rd2 and Abca4 -/- mice). The DHFR DD was effectively degraded in all model systems, including rd2 mice, which showed significant defects in chymotrypsin proteasomal activity. Moreover, trimethoprim (TMP) administration stabilized the DHFR DD in all mouse models. Thus, the DHFR DD-based approach allows for control of protein abundance in a variety of mouse models, laying the foundation to use this strategy for the conditional control of gene therapies to potentially treat multiple eye diseases.
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PURPOSE: A lack of sufficient functional information exists for appropriately categorizing a large number of myocilin (MYOC) variants and their involvement in primary open angle glaucoma, hindering their clinical significance classification. Most glaucoma-causing MYOC mutations result in protein non-secretion and intracellular insoluble aggregate formation in cultured cells. Herein, we generated a Gaussia luciferase-based MYOC fusion protein to quickly and sensitively track the secretion of MYOC variants and compared these results to the better-established western blotting assay for MYOC. METHODS: Fourteen clinically-derived MYOC variants with varying degrees of predicted pathogenicity were transfected into HEK-293A cells and analyzed by either a luciferase assay or western blotting. RESULTS: Eight of the variants (G12R, V53A, T204T, P254L, T325T, D380H, D395_E396insDP, and P481S) had not been biochemically assessed previously. Of these, P254L and D395_E396insDP demonstrated significant secretion defects reminiscent of glaucoma-causing mutations. The luciferase assay results agreed with western blotting for thirteen of the fourteen variants (93%), suggesting a strong concordance. CONCLUSIONS: These results suggest that the Gaussia luciferase assay may be used as a complementary or standalone assay for quickly assessing MYOC variant behavior and we anticipate that these results will be useful in MYOC variant curation and reclassification.
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Glaucoma de Ángulo Abierto , Glaucoma , Proteínas del Citoesqueleto , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Glaucoma de Ángulo Abierto/diagnóstico , Glaucoma de Ángulo Abierto/genética , Glaucoma de Ángulo Abierto/metabolismo , Glicoproteínas , Humanos , Luciferasas/genética , MutaciónRESUMEN
The antibiotic trimethoprim (TMP) is used to treat a variety of Escherichia coli infections, but its efficacy is limited by the rapid emergence of TMP-resistant bacteria. Previous laboratory evolution experiments have identified resistance-conferring mutations in the gene encoding the TMP target, bacterial dihydrofolate reductase (DHFR), in particular mutation L28R. Here, we show that 4'-desmethyltrimethoprim (4'-DTMP) inhibits both DHFR and its L28R variant, and selects against the emergence of TMP-resistant bacteria that carry the L28R mutation in laboratory experiments. Furthermore, antibiotic-sensitive E. coli populations acquire antibiotic resistance at a substantially slower rate when grown in the presence of 4'-DTMP than in the presence of TMP. We find that 4'-DTMP impedes evolution of resistance by selecting against resistant genotypes with the L28R mutation and diverting genetic trajectories to other resistance-conferring DHFR mutations with catalytic deficiencies. Our results demonstrate how a detailed characterization of resistance-conferring mutations in a target enzyme can help identify potential drugs against antibiotic-resistant bacteria, which may ultimately increase long-term efficacy of antimicrobial therapies by modulating evolutionary trajectories that lead to resistance.
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Infecciones por Escherichia coli/tratamiento farmacológico , Infecciones por Escherichia coli/microbiología , Resistencia al Trimetoprim/genética , Trimetoprim/análogos & derivados , Sustitución de Aminoácidos , Antibacterianos/química , Antibacterianos/farmacología , Cristalografía por Rayos X , Evolución Molecular Dirigida , Diseño de Fármacos , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Antagonistas del Ácido Fólico/química , Antagonistas del Ácido Fólico/farmacología , Genes Bacterianos , Genotipo , Humanos , Modelos Moleculares , Mutación , Tetrahidrofolato Deshidrogenasa/química , Tetrahidrofolato Deshidrogenasa/genética , Trimetoprim/química , Trimetoprim/farmacologíaRESUMEN
Purpose: Retinitis pigmentosa (RP) is an inherited retinal disorder that results in the degeneration of photoreceptor cells, ultimately leading to severe visual impairment. We characterized a consanguineous family from Southern India wherein a 25 year old individual presented with night blindness since childhood. The purpose of this study was to identify the causative mutation for RP in this individual as well as characterize how the mutation may ultimately affect protein function. Methods: We performed a complete ophthalmologic examination of the proband followed by exome sequencing. The likely causative mutation was identified and modeled in cultured cells, evaluating its expression, solubility (both with western blotting), subcellular distribution, (confocal microscopy), and testing whether this variant induced endoplasmic reticulum (ER) stress (quantitative PCR [qPCR] and western blotting). Results: The proband presented with generalized and parafoveal retinal pigmented epithelium (RPE) atrophy with bone spicule-like pigmentation in the midperiphery and arteriolar attenuation. Optical coherence tomography scans through the macula of both eyes showed atrophy of the outer retinal layers with loss of the ellipsoid zone, whereas the systemic examination of this individual was normal. The proband's parents and sibling were asymptomatic and had normal funduscopic examinations. We discovered a novel homozygous p.Pro388Ser mutation in the tubby-like protein 1 (TULP1) gene in the individual with RP. In cultured cells, the P388S mutation does not alter the subcellular distribution of TULP1 or induce ER stress when compared to wild-type TULP1, but instead significantly lowers protein stability as indicated with steady-state and cycloheximide-chase experiments. Conclusions: These results add to the list of known mutations in TULP1 identified in individuals with RP and suggest a possible unique pathogenic mechanism in TULP1-induced RP, which may be shared among select mutations in TULP1.
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Proteínas del Ojo/genética , Mutación Missense/genética , Retinitis Pigmentosa/genética , Adulto , Western Blotting , Consanguinidad , Electrorretinografía , Homocigoto , Humanos , India , Masculino , Microscopía Confocal , Linaje , Reacción en Cadena en Tiempo Real de la Polimerasa , Retinitis Pigmentosa/diagnóstico , Retinitis Pigmentosa/fisiopatología , Tomografía de Coherencia Óptica , Agudeza Visual/fisiología , Secuenciación del ExomaRESUMEN
Distinct mutations in the secreted extracellular matrix protein, fibulin-3 (F3), have been associated with a number of ocular diseases ranging from primary open angle glaucoma to cuticular age-related macular degeneration to a rare macular dystrophy, Malattia Leventinese (ML). The R345W F3 mutation that causes ML leads to F3 misfolding, inefficient secretion and accumulation at higher intracellular steady state levels in cultured cells. Herein, we determined whether fifteen other clinically-identified F3 mutations also led to similar levels of misfolding and secretion defects, which might provide insight into their potential pathogenicity. Surprisingly, we found that only a single F3 variant, L451F, presented with a significant secretion defect (69.5 ± 2.4% of wild-type (WT) F3 levels) and a corresponding increase in intracellular levels (226.8 ± 25.4% of WT F3 levels). Upon follow-up studies, when this conserved residue (L451) was mutated to a charged (Asp or Arg) or bulky (Pro, Trp, Tyr) residue, F3 secretion was also compromised, indicating the importance of small side chains (Leu, Ala, or Gly) at this residue. To uncover potential inherent F3 instability not easily observed under typical culture conditions, we genetically eliminated the sole stabilizing N-linked glycosylation site (N249) from select clinically-identified F3 mutants. This removal exacerbated R345W and L451F secretion defects (19.8 ± 3.0% and 12.4 ± 1.2% of WT F3 levels, respectively), but also revealed a previously undiscovered secretion defect in another C-terminal variant, Y397H (42.0 ± 10.1% of WT F3 levels). Yet, glycan removal did not change the relative secretion of the N-terminal mutants tested (D49A, R140W, I220F). These results highlight the uniqueness and molecular similarities between the R345W and L451F variants and also suggest that previously identified disease-associated mutations (e.g., R140W) are indistinguishable from WT with respect to secretion, hinting that they may lead to disease by an alternative mechanism.
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Proteínas de la Matriz Extracelular/genética , Glaucoma de Ángulo Abierto/genética , Degeneración Macular/genética , Línea Celular , Proteínas de la Matriz Extracelular/ultraestructura , Glaucoma de Ángulo Abierto/patología , Humanos , Degeneración Macular/patología , Mutación/genética , Drusas del Disco Óptico/congénito , Drusas del Disco Óptico/genética , Drusas del Disco Óptico/patología , Pliegue de Proteína , Estabilidad Proteica , Retina/metabolismo , Retina/patologíaRESUMEN
Purpose: Fuchs' endothelial corneal dystrophy (FECD) is the leading indication for corneal transplantation. Seventy percent of cases are caused by an intronic CTG triplet repeat expansion in the TCF4 gene that results in accumulation of pathogenic expanded CUG repeat RNA (CUGexp) as nuclear foci in corneal endothelium. A catalytically dead Cas9 (dCas9) can serve as an effective guide to target genomic DNA or RNA transcripts. Here, we examined the utility of the clustered regularly interspaced short palindromic repeats (CRISPR)-dCas9 system to effectively target and reduce CUGexp. Methods: We delivered dCas9 and repeat-targeting single guide RNA (sgRNA) expression plasmids to patient-derived endothelial cells using lipofection or lentiviral transduction. We used fluorescence in situ hybridization (FISH) and RNA dot-blot hybridization to quantify CUGexp foci and repeat RNA levels, respectively. TCF4 expression levels were assessed using quantitative PCR (qPCR). Results: Using FISH, we found that expression of both dCas9 and a (CAG)n sgRNA complementary to CUGexp are necessary to reduce foci. We observed a reduction in percentage of cells with foci from 59% to 5.6% and number of foci per 100 cells from 73.4 to 7.45 (P < 0.001) in cells stably expressing dCas9-(CAG)n sgRNA but saw no decrease in cells expressing dCas9-(CUG)n sgRNA or nontargeting control sgRNA. In cells with dCas9-(CAG)n sgRNA, we detected a reduction in CUGexp RNA by dot-blot without any reduction in TCF4 mRNA levels using qPCR. Conclusions: Using CRISPR-dCas9 to target the trinucleotide repeat is a promising treatment for FECD contingent on effective in vivo delivery. Translational Relevance: This work advances a gene therapy for a common age-related degenerative disorder.
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Células Endoteliales , Distrofia Endotelial de Fuchs , Distrofia Endotelial de Fuchs/genética , Humanos , Hibridación Fluorescente in Situ , Factor de Transcripción 4/genética , Expansión de Repetición de Trinucleótido/genéticaRESUMEN
The use of destabilizing domains (DDs) to conditionally control the abundance of a protein of interest (POI) through a small-molecule stabilizer has gained increasing traction both in vitro and in vivo. Yet there are specific considerations for the development and accurate control of user-defined POIs via DDs, as well as the identification of novel (and potentially synergistic) small-molecule stabilizers. Here, we describe a platform for achieving these goals. For complete details on the use and execution of this protocol, please refer to Ramadurgum et al. (2020).
Asunto(s)
Clonación Molecular/métodos , Proteínas Recombinantes , Línea Celular , Escherichia coli/genética , Células HEK293 , Humanos , Dominios Proteicos/genética , Ingeniería de Proteínas , Estabilidad Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Destabilizing domains (DDs) have been used successfully to conditionally control the abundance of proteins of interest (POIs) in a small-molecule-dependent manner in mice, worms (Caenorhabditis elegans), and Drosophila. However, development of such systems must account for delivery of the DD-POIs to the target tissue, accessibility of the target tissue to the small molecule, and quantification of stabilization. Here, we describe the considerations and steps to take in order to effectively implement a DD-POI in mouse ocular and hepatic tissue. For complete details on the use and execution of this protocol, please refer to Datta et al. (2018), Ramadurgum and Hulleman (2020), and Ramadurgum et al. (2020).