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Muna I Naash - One of the best experts on this subject based on the ideXlab platform.
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Syntaxin 3 is essential for photoreceptor outer segment protein trafficking and survival.
Proceedings of the National Academy of Sciences of the United States of America, 2020Co-Authors: Mashal Kakakhel, Shannon M Conley, Mustafa S Makia, Muayyad R. Al-ubaidi, Lars Tebbe, David M. Sherry, Muna I NaashAbstract:Trafficking of photoreceptor membrane proteins from their site of synthesis in the inner segment (IS) to the outer segment (OS) is critical for photoreceptor function and vision. Here we evaluate the role of syntaxin 3 (STX3), in trafficking of OS membrane proteins such as peripherin 2 (PRPH2) and rhodopsin. Photoreceptor-specific Stx3 knockouts [Stx3f/f(iCre75) and Stx3f/f(CRX-Cre)] exhibited rapid, early-onset photoreceptor degeneration and functional decline characterized by structural defects in IS, OS, and synaptic terminals. Critically, in the absence of STX3, OS proteins such as PRPH2, the PRPH2 binding partner, rod outer segment membrane protein 1 (ROM1), and rhodopsin were mislocalized along the microtubules to the IS, cell body, and synaptic region. We find that the PRPH2 C-terminal domain interacts with STX3 as well as other photoreceptor SNAREs, and our findings indicate that STX3 is an essential part of the trafficking pathway for both disc (rhodopsin) and rim (PRPH2/ROM1) components of the OS.
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ROM1 contributes to phenotypic heterogeneity in PRPH2-associated retinal disease.
Human molecular genetics, 2020Co-Authors: Daniel G. Strayve, Shannon M Conley, Mustafa S Makia, Mashal Kakakhel, Haarthi Sakthivel, Muayyad R. Al-ubaidi, Muna I NaashAbstract:Peripherin 2 (PRPH2) is a retina-specific tetraspanin protein essential for the formation of rod and cone photoreceptor outer segments (OS). Patients with mutations in PRPH2 exhibit severe retinal degeneration characterized by vast inter- and intra-familial phenotypic heterogeneity. To help understand contributors to this within-mutation disease variability, we asked whether the PRPH2 binding partner rod OS membrane protein 1 (ROM1) could serve as a phenotypic modifier. We utilized knockin and transgenic mouse models to evaluate the structural, functional and biochemical effects of eliminating one allele of ROM1 (ROM1+/-) in three different Prph2 models which mimic human disease: C213Y Prph2 (Prph2C/+), K153Del Prph2 (Prph2K/+) and R172W (Prph2R172W). Reducing ROM1 in the absence of Prph2 mutations (ROM1+/-) had no effect on retinal structure or function. However, the effects of reducing ROM1 in the presence of Prph2 mutations were highly variable. Prph2K/+/ROM1+/- mice had improved rod and cone function compared with Prph2K/+ as well as amelioration of K153Del-associated defects in PRPH2/ROM1 oligomerization. In contrast, Prph2R172W/ROM1+/- animals had worsened rod and cone function and exacerbated retinal degeneration compared with Prph2R172W animals. Removing one allele of ROM1 had no effect in Prph2C/+. Combined, our findings support a role for non-pathogenic ROM1 null variants in contributing to phenotypic variability in mutant PRPH2-associated retinal degeneration. Since the effects of ROM1 reduction are variable, our data suggest that this contribution is specific to the type of Prph2 mutation.
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Prph2 initiates outer segment morphogenesis but maturation requires Prph2/ROM1 oligomerization.
Human molecular genetics, 2019Co-Authors: Shannon M Conley, Rahel Zulliger, Michael W. Stuck, Jamie N. Watson, Justin L. Burnett, Muna I NaashAbstract:The retinal disease gene peripherin 2 (PRPH2) is essential for the formation of photoreceptor outer segments (OSs), where it functions in oligomers with and without its homologue ROM1. However, the precise role of these proteins in OS morphogenesis is not understood. By utilizing a knock-in mouse expressing a chimeric protein comprised of the body of ROM1 and the C-terminus of Prph2 (termed RRCT), we find that the Prph2 C-terminus is necessary and sufficient for the initiation of OSs, while OS maturation requires the body of Prph2 and associated large oligomers. Importantly, dominant-negative physiological and biochemical defects in RRCT heterozygous rods are rescued by removing ROM1, suggesting ROM1 is a regulator for OS formation. Our experiments evaluating Prph2 trafficking show that ROM1 is a key determinant of whether Prph2 complexes utilize conventional versus unconventional (Golgi bypass) secretory pathways to reach the OS. These findings significantly advance our understanding of the molecular underpinnings of OS morphogenesis and particularly the role of ROM1.
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oligomerization of prph2 and ROM1 is essential for photoreceptor outer segment formation
Human Molecular Genetics, 2018Co-Authors: Rahel Zulliger, Muayyad R Alubaidi, Shannon M Conley, Maggie L Mwoyosvi, Muna I NaashAbstract:Mutations in peripherin 2 (PRPH2, also known as Rds), a tetraspanin protein found in photoreceptor outer segments (OSs), cause retinal degeneration ranging from rod-dominant retinitis pigmentosa (RP) to cone-dominant macular dystrophy (MD). Understanding why some Prph2 mutants affect rods while others affect cones remains a critical unanswered question. Prph2 is essential for OS structure and function and exhibits a very specific pattern of oligomerization with its homolog ROM1. Non-covalent Prph2/ROM1 homo- and hetero-tetramers assemble into higher-order covalently linked complexes held together by an intermolecular disulfide bond at Prph2-C150/ROM1-C153. Here we disrupt this crucial bond using a C150S-Prph2 knockin mouse line to study the role of Prph2 higher-order complex formation. We find that C150S-Prph2 traffics to the OS, interacts with ROM1 and forms non-covalent tetramers, but alone cannot support normal OS structure and function. However, C150S-Prph2 supports the initiation or elaboration of OS disc structures, and improves rod OS ultrastructure in the presence of wild-type (WT) Prph2 (i.e. Prph2C150S/+ versus Prph2+/−). Prph2C150S/+ animals exhibit haploinsufficiency in rods, but a dominant-negative phenotype in cones, suggesting cones have a different requirement for large Prph2 complexes than rods. Importantly, cone but not rod function can be improved by the addition of one Prph2Y141C allele, a mutation responsible for pattern dystrophy owing to the extra cysteine. Combined these findings show that covalently linked Prph2 complexes are essential for OS formation, but not for Prph2 targeting to the OS, and that cones are especially sensitive to having a broad distribution of Prph2 complex types (i.e. tetramers and large complexes).
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prph2 initiates outer segment morphogenesis but maturation requires prph2 ROM1 oligomerization
Human Molecular Genetics, 2018Co-Authors: Shannon M Conley, Rahel Zulliger, Michael W. Stuck, Jamie N. Watson, Justin L. Burnett, Muna I NaashAbstract:The retinal disease gene peripherin 2 (PRPH2) is essential for the formation of photoreceptor outer segments (OSs), where it functions in oligomers with and without its homologue ROM1. However, the precise role of these proteins in OS morphogenesis is not understood. By utilizing a knock-in mouse expressing a chimeric protein comprised of the body of ROM1 and the C-terminus of Prph2 (termed RRCT), we find that the Prph2 C-terminus is necessary and sufficient for the initiation of OSs, while OS maturation requires the body of Prph2 and associated large oligomers. Importantly, dominant-negative physiological and biochemical defects in RRCT heterozygous rods are rescued by removing ROM1, suggesting ROM1 is a regulator for OS formation. Our experiments evaluating Prph2 trafficking show that ROM1 is a key determinant of whether Prph2 complexes utilize conventional versus unconventional (Golgi bypass) secretory pathways to reach the OS. These findings significantly advance our understanding of the molecular underpinnings of OS morphogenesis and particularly the role of ROM1.
Gabriel H. Travis - One of the best experts on this subject based on the ideXlab platform.
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Deficiency of rds/peripherin causes photoreceptor death in mouse models of digenic and dominant retinitis pigmentosa.
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: Wojciech Kedzierski, Roderick R. Mcinnes, Steven Nusinowitz, David G. Birch, Geoff Clarke, Dean Bok, Gabriel H. TravisAbstract:Retinitis pigmentosa (RP) is a group of inherited blinding diseases caused by mutations in multiple genes including RDS. RDS encodes rds/peripherin (rds), a 36-kDa glycoprotein in the rims of rod and cone outer-segment (OS) discs. ROM1 is related to rds with similar membrane topology and the identical distribution in OS. In contrast to RDS, no mutations in ROM1 alone have been associated with retinal disease. However, an unusual digenic form of RP has been described. Affected individuals in several families were doubly heterozygous for a mutation in RDS causing a leucine 185 to proline substitution in rds (L185P) and a null mutation in ROM1. Neither mutation alone caused clinical abnormalities. Here, we generated transgenic/knockout mice that duplicate the amino acid substitutions and predicted levels of rds and ROM1 in patients with RDS-mediated digenic and dominant RP. Photoreceptor degeneration in the mouse model of digenic RP was faster than in the wild-type and monogenic controls by histological, electroretinographic, and biochemical analysis. We observed a positive correlation between the rate of photoreceptor loss and the extent of OS disorganization in mice of several genotypes. Photoreceptor degeneration in RDS-mediated RP appears to be caused by a simple deficiency of rds and ROM1. The critical threshold for the combined abundance of rds and ROM1 is ≈60% of wild type. Below this value, the extent of OS disorganization results in clinically significant photoreceptor degeneration.
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Analysis of the rds/peripherin.ROM1 complex in transgenic photoreceptors that express a chimeric protein.
The Journal of biological chemistry, 1999Co-Authors: Wojciech Kedzierski, Jian Weng, Gabriel H. TravisAbstract:Abstract Mice homozygous for the retinal degeneration slow (rds) mutation completely lack photoreceptor outer segments. The rds gene encodes rds/peripherin (rds), a membrane glycoprotein in the rims of rod and cone outer segment discs. rds is present as a complex with the related protein, ROM1. Here, we generated transgenic mice that express a chimeric protein (rom/D2) containing the intradiscal D2 loop of rds in the context of ROM1. rom/D2 was N-glycosylated, formed covalent homodimers, and interacted non-covalently with itself, rds, and ROM1. The rds·rom/D2 interaction was significantly more stable than the non-covalent interaction between rds and ROM1 by detergent/urea titration. Analysis of mice expressing rom/D2 revealed that rds is 2.5-fold more abundant than ROM1, interacts non-covalently with itself and ROM1 via the D2 loop, and forms a high order complex that may extend the entire circumference of the disc. Expression of rom/D2 fully rescued the ultrastructural phenotype inrds+/− mutant mice, but it had no effect on the phenotype in rds−/− mutants. Together, these observations explain the striking differences in null phenotypes and frequencies of disease-causing mutations between the RDS andROM1 genes.
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analysis of the rds peripherin ROM1 complex in transgenic photoreceptors that express a chimeric protein
Journal of Biological Chemistry, 1999Co-Authors: Wojciech Kedzierski, Jian Weng, Gabriel H. TravisAbstract:Abstract Mice homozygous for the retinal degeneration slow (rds) mutation completely lack photoreceptor outer segments. The rds gene encodes rds/peripherin (rds), a membrane glycoprotein in the rims of rod and cone outer segment discs. rds is present as a complex with the related protein, ROM1. Here, we generated transgenic mice that express a chimeric protein (rom/D2) containing the intradiscal D2 loop of rds in the context of ROM1. rom/D2 was N-glycosylated, formed covalent homodimers, and interacted non-covalently with itself, rds, and ROM1. The rds·rom/D2 interaction was significantly more stable than the non-covalent interaction between rds and ROM1 by detergent/urea titration. Analysis of mice expressing rom/D2 revealed that rds is 2.5-fold more abundant than ROM1, interacts non-covalently with itself and ROM1 via the D2 loop, and forms a high order complex that may extend the entire circumference of the disc. Expression of rom/D2 fully rescued the ultrastructural phenotype inrds+/− mutant mice, but it had no effect on the phenotype in rds−/− mutants. Together, these observations explain the striking differences in null phenotypes and frequencies of disease-causing mutations between the RDS andROM1 genes.
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Transgenic analysis of rds/peripherin N-glycosylation: effect on dimerization, interaction with ROM1, and rescue of the rds null phenotype.
Journal of neurochemistry, 1999Co-Authors: Wojciech Kedzierski, Dean Bok, Gabriel H. TravisAbstract:Abstract : Rds/peripherin is an integral membrane glycoprotein that is present in the rims of photoreceptor outer segment disks. In mammals, it is thought to stabilize the disk rim through heterophilic interactions with the related nonglycosylated protein ROM1. Glycosylation of rds/peripherin at asparagine 229 is widely conserved in vertebrates. In this study, we investigated the role of rds/peripherin N-glycosylation. We generated transgenic mice that expressed only S231A-substituted rds/peripherin in their retinas. This protein was not glycosylated but formed covalent dimers with itself and with glycosylated rds/peripherin. Nonglycosylated rds/peripherin also interacted noncovalently with ROM1 homodimers to form a heterooligomeric complex. The glycosylated rds/peripherin ·· ROM1 complex bound to concanavalin A-Sepharose, suggesting that the glycan is not directly involved in the interaction between these proteins. In double transgenic mice expressing normal and S231A-substituted rds/peripherin, the mRNA-to-protein ratios were similar for both transgenes, indicating no effect of N-glycosylation on rds/peripherin stability. Finally, expression of nonglycosylated rds/peripherin in transgenic mice rescued the phenotype of outer segment nondevelopment in retinal degeneration slow (rds-/-) null mutants. These observations indicate that N-glycosylation of rds/peripherin is not required for its normal processing, stability, or in vivo function.
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transgenic analysis of rds peripherin n glycosylation effect on dimerization interaction with ROM1 and rescue of the rds null phenotype
Journal of Neurochemistry, 1999Co-Authors: Wojciech Kedzierski, Dean Bok, Gabriel H. TravisAbstract:Abstract : Rds/peripherin is an integral membrane glycoprotein that is present in the rims of photoreceptor outer segment disks. In mammals, it is thought to stabilize the disk rim through heterophilic interactions with the related nonglycosylated protein ROM1. Glycosylation of rds/peripherin at asparagine 229 is widely conserved in vertebrates. In this study, we investigated the role of rds/peripherin N-glycosylation. We generated transgenic mice that expressed only S231A-substituted rds/peripherin in their retinas. This protein was not glycosylated but formed covalent dimers with itself and with glycosylated rds/peripherin. Nonglycosylated rds/peripherin also interacted noncovalently with ROM1 homodimers to form a heterooligomeric complex. The glycosylated rds/peripherin ·· ROM1 complex bound to concanavalin A-Sepharose, suggesting that the glycan is not directly involved in the interaction between these proteins. In double transgenic mice expressing normal and S231A-substituted rds/peripherin, the mRNA-to-protein ratios were similar for both transgenes, indicating no effect of N-glycosylation on rds/peripherin stability. Finally, expression of nonglycosylated rds/peripherin in transgenic mice rescued the phenotype of outer segment nondevelopment in retinal degeneration slow (rds-/-) null mutants. These observations indicate that N-glycosylation of rds/peripherin is not required for its normal processing, stability, or in vivo function.
Roger P. Wise - One of the best experts on this subject based on the ideXlab platform.
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mla and ROM1 mediated control of microrna398 and chloroplast copper zinc superoxide dismutase regulates cell death in response to the barley powdery mildew fungus
New Phytologist, 2014Co-Authors: Yan Meng, Roger P. WiseAbstract:• Barley (Hordeum vulgare L.) Mildew resistance locus a (Mla) confers allele-specific interactions with natural variants of the ascomycete fungus Blumeria graminis f. sp. hordei (Bgh), the causal agent of powdery mildew disease. Significant reprogramming of Mla-mediated gene expression occurs upon infection by this obligate biotrophic pathogen. • We utilized a proteomics-based approach, combined with barley mla, required for Mla12 resistance1 (rar1), and restoration of Mla resistance1 (ROM1) mutants, to identify components of Mla-directed signaling. • Loss-of-function mutations in Mla and Rar1 both resulted in the reduced accumulation of chloroplast copper/zinc superoxide dismutase 1 (HvSOD1), whereas loss of function in ROM1 re-established HvSOD1 levels. In addition, both Mla and ROM1 negatively regulated hvu-microRNA398 (hvu-miR398), and up-regulation of miR398 was coupled to reduced HvSOD1 expression. Barley stripe mosaic virus (BSMV)-mediated over-expression of both barley and Arabidopsis miR398 repressed accumulation of HvSOD1, and BSMV-induced gene silencing of HvSod1 impeded Mla-triggered H₂O₂ and hypersensitive reaction (HR) at barley-Bgh interaction sites. • These data indicate that Mla- and ROM1-regulated hvu-miR398 represses HvSOD1 accumulation, influencing effector-induced HR in response to the powdery mildew fungus.
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Mla- and ROM1-mediated control of microRNA398 and chloroplast copper/zinc superoxide dismutase regulates cell death in response to the barley powdery mildew fungus
The New phytologist, 2013Co-Authors: Yan Meng, Roger P. WiseAbstract:• Barley (Hordeum vulgare L.) Mildew resistance locus a (Mla) confers allele-specific interactions with natural variants of the ascomycete fungus Blumeria graminis f. sp. hordei (Bgh), the causal agent of powdery mildew disease. Significant reprogramming of Mla-mediated gene expression occurs upon infection by this obligate biotrophic pathogen. • We utilized a proteomics-based approach, combined with barley mla, required for Mla12 resistance1 (rar1), and restoration of Mla resistance1 (ROM1) mutants, to identify components of Mla-directed signaling. • Loss-of-function mutations in Mla and Rar1 both resulted in the reduced accumulation of chloroplast copper/zinc superoxide dismutase 1 (HvSOD1), whereas loss of function in ROM1 re-established HvSOD1 levels. In addition, both Mla and ROM1 negatively regulated hvu-microRNA398 (hvu-miR398), and up-regulation of miR398 was coupled to reduced HvSOD1 expression. Barley stripe mosaic virus (BSMV)-mediated over-expression of both barley and Arabidopsis miR398 repressed accumulation of HvSOD1, and BSMV-induced gene silencing of HvSod1 impeded Mla-triggered H₂O₂ and hypersensitive reaction (HR) at barley-Bgh interaction sites. • These data indicate that Mla- and ROM1-regulated hvu-miR398 represses HvSOD1 accumulation, influencing effector-induced HR in response to the powdery mildew fungus.
Roderick R. Mcinnes - One of the best experts on this subject based on the ideXlab platform.
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Deficiency of rds/peripherin causes photoreceptor death in mouse models of digenic and dominant retinitis pigmentosa.
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: Wojciech Kedzierski, Roderick R. Mcinnes, Steven Nusinowitz, David G. Birch, Geoff Clarke, Dean Bok, Gabriel H. TravisAbstract:Retinitis pigmentosa (RP) is a group of inherited blinding diseases caused by mutations in multiple genes including RDS. RDS encodes rds/peripherin (rds), a 36-kDa glycoprotein in the rims of rod and cone outer-segment (OS) discs. ROM1 is related to rds with similar membrane topology and the identical distribution in OS. In contrast to RDS, no mutations in ROM1 alone have been associated with retinal disease. However, an unusual digenic form of RP has been described. Affected individuals in several families were doubly heterozygous for a mutation in RDS causing a leucine 185 to proline substitution in rds (L185P) and a null mutation in ROM1. Neither mutation alone caused clinical abnormalities. Here, we generated transgenic/knockout mice that duplicate the amino acid substitutions and predicted levels of rds and ROM1 in patients with RDS-mediated digenic and dominant RP. Photoreceptor degeneration in the mouse model of digenic RP was faster than in the wild-type and monogenic controls by histological, electroretinographic, and biochemical analysis. We observed a positive correlation between the rate of photoreceptor loss and the extent of OS disorganization in mice of several genotypes. Photoreceptor degeneration in RDS-mediated RP appears to be caused by a simple deficiency of rds and ROM1. The critical threshold for the combined abundance of rds and ROM1 is ≈60% of wild type. Below this value, the extent of OS disorganization results in clinically significant photoreceptor degeneration.
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Digenic inheritance of a ROM1 gene mutation with a peripherin/RDS or rhodopsin mutation in families with retinitis pigmentosa
1999Co-Authors: Samuel G. Jacobson, Artur V. Cideciyan, Roger A. Bascom, Vesna Ponjavic, Magnus Abrahamson, Ulf Ekström, Sten Andréasson, Berndt Ehinger, Val C. Sheffield, Roderick R. McinnesAbstract:Two families with retinitis pigmentosa showed inheritance of an Arg-16-His ROM1 gene mutation with either an Arg-13-Trp RDS mutation or an Arg-135-Trp RHO mutation. The phenotypes of double and single heterozygotes were determined to examine the hypothesis that digenic inheritance may increase disease expression. In the family with ROM1 and RDS mutations, single heterozygotes were normal but one double heterozygote showed severe RP. Two other double heterozygotes, however, were normal by clinical and retinal function tests. In the family with ROM1 and RHO mutations, single heterozygotes with the RHO mutation all manifested RP, while a single heterozygote for the ROM1 mutation was normal. Disease severity was comparable in double heterozygotes and single heterozygotes HAVING the RHO mutation. We conclude that the Arg-16-His ROM1 gene mutation is non-pathogenic in the single heterozygous state, and there is no consistent evidence of digenic augmentation of pathogenicity in double heterozygotes carrying the Arg-16-His ROM1 mutation with either the benign Arg-13-Trp RDS mutation or the disease-causing Arg-135-Trp RHO mutation. (Less)
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digenic inheritance of a ROM1 gene mutation with a peripherin rds or rhodopsin mutation in families with retinitis pigmentosa
Digital Journal of Ophthalmology; 5(6) (1999), 1999Co-Authors: Samuel G. Jacobson, Artur V. Cideciyan, Roger A. Bascom, Vesna Ponjavic, Magnus Abrahamson, Ulf Ekström, Sten Andréasson, Berndt Ehinger, Val C. Sheffield, Roderick R. McinnesAbstract:Two families with retinitis pigmentosa showed inheritance of an Arg-16-His ROM1 gene mutation with either an Arg-13-Trp RDS mutation or an Arg-135-Trp RHO mutation. The phenotypes of double and single heterozygotes were determined to examine the hypothesis that digenic inheritance may increase disease expression. In the family with ROM1 and RDS mutations, single heterozygotes were normal but one double heterozygote showed severe RP. Two other double heterozygotes, however, were normal by clinical and retinal function tests. In the family with ROM1 and RHO mutations, single heterozygotes with the RHO mutation all manifested RP, while a single heterozygote for the ROM1 mutation was normal. Disease severity was comparable in double heterozygotes and single heterozygotes HAVING the RHO mutation. We conclude that the Arg-16-His ROM1 gene mutation is non-pathogenic in the single heterozygous state, and there is no consistent evidence of digenic augmentation of pathogenicity in double heterozygotes carrying the Arg-16-His ROM1 mutation with either the benign Arg-13-Trp RDS mutation or the disease-causing Arg-135-Trp RHO mutation. (Less)
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Mutation analysis of the ROM1 gene in retinitis pigmentosa
Human molecular genetics, 1995Co-Authors: Roger A. Bascom, Lin Liu, John R. Heckenlively, Edwin M. Stone, Roderick R. McinnesAbstract:To examine the role of ROM1, a homologue of peripherin/RDS, in autosomal dominant retinitis pigmentosa (adRP), we screened 224 adRP and 29 simplex RP probands for ROM1 mutations. Four ROM1 alleles were designated as potentially pathogenic because they were found only in RP patients but not in 50-100 controls nor in 249 other RP probands. The substitutions P60T and T108M were present in a single allele in a subject with typical adRP, and this allele cosegregated with the disease in the small family. The putative null allele L114 [1 bp] was present in an individual with atypical RP but not in three unaffected siblings. This insertion has been previously reported to cause RP only when accompanied by a peripherin/RDS mutation, but no peripherin/RDS mutations were found in any of the four probands reported here. Two substitutions (G75D, R242Q) were present in two other probands with simplex RP. These data suggest that potentially pathogenic ROM1 mutations occur in 1% or less of patients with adRP or simplex RP. The absence of detectable peripherin/RDS mutations in these families suggests either that: (i) mutations in other digenic partners are required for pathogenic ROM1 alleles to cause retinal degeneration; (ii) these ROM1 mutations do not cause RP; or (iii) peripherin/RDS mutations are present but were not identified in these patients.
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Refining the locus for Best vitelliform macular dystrophy and mutation analysis of the candidate gene ROM1.
American journal of human genetics, 1994Co-Authors: Brian E. Nichols, Roger A. Bascom, Val C. Sheffield, Roderick R. Mcinnes, M. Litt, Edwin M. StoneAbstract:Vitelliform macular dystrophy (Best disease) is an autosomal dominant macular dystrophy which shares important clinical features with age-related macular degeneration, the most common cause of legal blindness in the elderly. Unfortunately, understanding and treatment for this common age-related disorder is limited. Discovery of the gene which causes Best disease has the potential to increase the understanding of the pathogenesis of all types of macular degeneration, including the common age-related form. Best disease has recently been mapped to chromosome 11q13. The photoreceptor-specific protein ROM1 has also been recently mapped to this location, and the ROM1 gene is a candidate gene for Best disease. Using highly polymorphic markers, the authors have narrowed the genetic region which contains the Best disease gene to the 10-cM region between markers D11S871 and PYGM. Marker D11S956 demonstrated no recombinants with Best disease in three large families and resulted in a lod score of 18.2. In addition, a polymorphism within the ROM1 gene also demonstrated no recombinants and resulted in a lod score of 10.0 in these same three families. The authors used a combination of SSCP analysis, denaturing gradient gel electrophoresis, and DNA sequencing to screen the entire coding region of the ROM1 gene in 11more » different unrelated patients affected with Best disease. No nucleotide changes were found in the coding sequence of any affected patient, indicating that mutations within the coding sequence are unlikely to cause Best disease. 28 refs., 3 figs., 2 tabs.« less
Wojciech Kedzierski - One of the best experts on this subject based on the ideXlab platform.
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Deficiency of rds/peripherin causes photoreceptor death in mouse models of digenic and dominant retinitis pigmentosa.
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: Wojciech Kedzierski, Roderick R. Mcinnes, Steven Nusinowitz, David G. Birch, Geoff Clarke, Dean Bok, Gabriel H. TravisAbstract:Retinitis pigmentosa (RP) is a group of inherited blinding diseases caused by mutations in multiple genes including RDS. RDS encodes rds/peripherin (rds), a 36-kDa glycoprotein in the rims of rod and cone outer-segment (OS) discs. ROM1 is related to rds with similar membrane topology and the identical distribution in OS. In contrast to RDS, no mutations in ROM1 alone have been associated with retinal disease. However, an unusual digenic form of RP has been described. Affected individuals in several families were doubly heterozygous for a mutation in RDS causing a leucine 185 to proline substitution in rds (L185P) and a null mutation in ROM1. Neither mutation alone caused clinical abnormalities. Here, we generated transgenic/knockout mice that duplicate the amino acid substitutions and predicted levels of rds and ROM1 in patients with RDS-mediated digenic and dominant RP. Photoreceptor degeneration in the mouse model of digenic RP was faster than in the wild-type and monogenic controls by histological, electroretinographic, and biochemical analysis. We observed a positive correlation between the rate of photoreceptor loss and the extent of OS disorganization in mice of several genotypes. Photoreceptor degeneration in RDS-mediated RP appears to be caused by a simple deficiency of rds and ROM1. The critical threshold for the combined abundance of rds and ROM1 is ≈60% of wild type. Below this value, the extent of OS disorganization results in clinically significant photoreceptor degeneration.
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Analysis of the rds/peripherin.ROM1 complex in transgenic photoreceptors that express a chimeric protein.
The Journal of biological chemistry, 1999Co-Authors: Wojciech Kedzierski, Jian Weng, Gabriel H. TravisAbstract:Abstract Mice homozygous for the retinal degeneration slow (rds) mutation completely lack photoreceptor outer segments. The rds gene encodes rds/peripherin (rds), a membrane glycoprotein in the rims of rod and cone outer segment discs. rds is present as a complex with the related protein, ROM1. Here, we generated transgenic mice that express a chimeric protein (rom/D2) containing the intradiscal D2 loop of rds in the context of ROM1. rom/D2 was N-glycosylated, formed covalent homodimers, and interacted non-covalently with itself, rds, and ROM1. The rds·rom/D2 interaction was significantly more stable than the non-covalent interaction between rds and ROM1 by detergent/urea titration. Analysis of mice expressing rom/D2 revealed that rds is 2.5-fold more abundant than ROM1, interacts non-covalently with itself and ROM1 via the D2 loop, and forms a high order complex that may extend the entire circumference of the disc. Expression of rom/D2 fully rescued the ultrastructural phenotype inrds+/− mutant mice, but it had no effect on the phenotype in rds−/− mutants. Together, these observations explain the striking differences in null phenotypes and frequencies of disease-causing mutations between the RDS andROM1 genes.
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analysis of the rds peripherin ROM1 complex in transgenic photoreceptors that express a chimeric protein
Journal of Biological Chemistry, 1999Co-Authors: Wojciech Kedzierski, Jian Weng, Gabriel H. TravisAbstract:Abstract Mice homozygous for the retinal degeneration slow (rds) mutation completely lack photoreceptor outer segments. The rds gene encodes rds/peripherin (rds), a membrane glycoprotein in the rims of rod and cone outer segment discs. rds is present as a complex with the related protein, ROM1. Here, we generated transgenic mice that express a chimeric protein (rom/D2) containing the intradiscal D2 loop of rds in the context of ROM1. rom/D2 was N-glycosylated, formed covalent homodimers, and interacted non-covalently with itself, rds, and ROM1. The rds·rom/D2 interaction was significantly more stable than the non-covalent interaction between rds and ROM1 by detergent/urea titration. Analysis of mice expressing rom/D2 revealed that rds is 2.5-fold more abundant than ROM1, interacts non-covalently with itself and ROM1 via the D2 loop, and forms a high order complex that may extend the entire circumference of the disc. Expression of rom/D2 fully rescued the ultrastructural phenotype inrds+/− mutant mice, but it had no effect on the phenotype in rds−/− mutants. Together, these observations explain the striking differences in null phenotypes and frequencies of disease-causing mutations between the RDS andROM1 genes.
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Transgenic analysis of rds/peripherin N-glycosylation: effect on dimerization, interaction with ROM1, and rescue of the rds null phenotype.
Journal of neurochemistry, 1999Co-Authors: Wojciech Kedzierski, Dean Bok, Gabriel H. TravisAbstract:Abstract : Rds/peripherin is an integral membrane glycoprotein that is present in the rims of photoreceptor outer segment disks. In mammals, it is thought to stabilize the disk rim through heterophilic interactions with the related nonglycosylated protein ROM1. Glycosylation of rds/peripherin at asparagine 229 is widely conserved in vertebrates. In this study, we investigated the role of rds/peripherin N-glycosylation. We generated transgenic mice that expressed only S231A-substituted rds/peripherin in their retinas. This protein was not glycosylated but formed covalent dimers with itself and with glycosylated rds/peripherin. Nonglycosylated rds/peripherin also interacted noncovalently with ROM1 homodimers to form a heterooligomeric complex. The glycosylated rds/peripherin ·· ROM1 complex bound to concanavalin A-Sepharose, suggesting that the glycan is not directly involved in the interaction between these proteins. In double transgenic mice expressing normal and S231A-substituted rds/peripherin, the mRNA-to-protein ratios were similar for both transgenes, indicating no effect of N-glycosylation on rds/peripherin stability. Finally, expression of nonglycosylated rds/peripherin in transgenic mice rescued the phenotype of outer segment nondevelopment in retinal degeneration slow (rds-/-) null mutants. These observations indicate that N-glycosylation of rds/peripherin is not required for its normal processing, stability, or in vivo function.
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transgenic analysis of rds peripherin n glycosylation effect on dimerization interaction with ROM1 and rescue of the rds null phenotype
Journal of Neurochemistry, 1999Co-Authors: Wojciech Kedzierski, Dean Bok, Gabriel H. TravisAbstract:Abstract : Rds/peripherin is an integral membrane glycoprotein that is present in the rims of photoreceptor outer segment disks. In mammals, it is thought to stabilize the disk rim through heterophilic interactions with the related nonglycosylated protein ROM1. Glycosylation of rds/peripherin at asparagine 229 is widely conserved in vertebrates. In this study, we investigated the role of rds/peripherin N-glycosylation. We generated transgenic mice that expressed only S231A-substituted rds/peripherin in their retinas. This protein was not glycosylated but formed covalent dimers with itself and with glycosylated rds/peripherin. Nonglycosylated rds/peripherin also interacted noncovalently with ROM1 homodimers to form a heterooligomeric complex. The glycosylated rds/peripherin ·· ROM1 complex bound to concanavalin A-Sepharose, suggesting that the glycan is not directly involved in the interaction between these proteins. In double transgenic mice expressing normal and S231A-substituted rds/peripherin, the mRNA-to-protein ratios were similar for both transgenes, indicating no effect of N-glycosylation on rds/peripherin stability. Finally, expression of nonglycosylated rds/peripherin in transgenic mice rescued the phenotype of outer segment nondevelopment in retinal degeneration slow (rds-/-) null mutants. These observations indicate that N-glycosylation of rds/peripherin is not required for its normal processing, stability, or in vivo function.
