The Experts below are selected from a list of 1863 Experts worldwide ranked by ideXlab platform
Lizbeth Hedstrom - One of the best experts on this subject based on the ideXlab platform.
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Mycophenolic anilides as broad specificity inosine-5’-monophosphate dehydrogenase (IMPDH) inhibitors
Bioorganic & medicinal chemistry letters, 2020Co-Authors: Seungheon Lee, Lizbeth Hedstrom, Minjia Zhang, Mohana Rao Vippila, Yong Wang, Xingyou Wang, Gregory D. CunyAbstract:Inosine-5'-monophosphate dehydrogenase (IMPDH) is a potential target for microorganisms. However, identifying inhibitor design determinants for IMPDH orthologs continues to evolve. Herein, a series of mycophenolic anilide inhibitors of Cryptosporidium parvum and human IMPDHs are reported. Furthermore, molecular docking of 12 (e.g. SH-19; CpIMPDH Ki,app = 0.042 ± 0.015 µM, HsIMPDH2 Ki,app = 0.13 ± 0.05 µM) supports different binding modes with the two enzymes. For CpIMPDH the inhibitor extends into a pocket in an adjacent subunit. In contrast, docking suggests the inhibitor interacts with Ser276 in the NAD binding site in HsIMPDH2, as well as an adjacent pocket within the same subunit. These results provide further guidance for generating IMPDH inhibitors for enzymes found in an array of pathogenic microorganisms, including Mycobacterium tuberculosis.
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A structural determinant of mycophenolic acid resistance in eukaryotic inosine 5'-monophosphate dehydrogenases.
Protein science : a publication of the Protein Society, 2019Co-Authors: Rebecca Freedman, Alexander W. Sarkis, Lizbeth HedstromAbstract:Mycophenolic acid (MPA) is a potent natural product inhibitor of fungal and other eukaryotic inosine 5'-monophosphate dehydrogenases (IMPDHs) originally isolated from spoiled corn silage. MPA is produced by the filamentous fungi Penicillium brevicompactum, which contains two IMPDHs, PbIMPDHA and PbIMPDHB, both of which are MPA-resistant. The MPA binding sites of these enzymes are identical to MPA-sensitive IMPDHs, so the structural determinants of resistance are unknown. Here we show that a single residue, Ser267, accounts for the MPA resistance of PbIMPDHA. Substitution of Ser267 with Ala, the residue most commonly found in this position in eukaryotic IMPDHs, makes PbIMPDHA sensitive to MPA. Conversely, Aspergillus nidulans IMPDH becomes MPA-resistant when the analogous Ala residue is substituted with Ser. These substitutions have little effect on the catalytic cycles of either enzyme, suggesting the fitness costs are negligible despite the strong conservation of Ala at this position. Intriguingly, while only 1% of fungal IMPDHs contain Ser or Thr at position 267, these residues are found in the IMPDHs from several Aspergillus species that grow at the low temperatures also favored by Penicillium. Perhaps Ser/Thr267 is an evolutionary signature of MPA exposure.
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Oxanosine Monophosphate Is a Covalent Inhibitor of Inosine 5′-Monophosphate Dehydrogenase
2019Co-Authors: Youngchang Kim, Natalia Maltseva, Philip Braunstein, Andrzej Joachimiak, Lizbeth HedstromAbstract:Reactive nitrogen species (RNS) are produced during infection and inflammation, and the effects of these agents on proteins, DNA, and lipids are well recognized. In contrast, the effects of RNS damaged metabolites are less appreciated. 5-Amino-3-β-(d-ribofuranosyl)-3 H-imidazo-[4,5-d][1,3]oxazine-7-one (oxanosine) and its nucleotides are products of guanosine nitrosation. Here we demonstrate that oxanosine monophosphate (OxMP) is a potent reversible competitive inhibitor of IMPDH. The value of Ki varies from 50 to 340 nM among IMPDHs from five different organisms. UV spectroscopy and X-ray crystallography indicate that OxMP forms a ring-opened covalent adduct with the active site Cys (E-OxMP*). Unlike the covalent intermediate of the normal catalytic reaction, E-OxMP* does not hydrolyze, but instead recyclizes to OxMP. IMPDH inhibitors block proliferation and can induce apoptosis, so the inhibition of IMPDH by OxMP presents another potential mechanism for RNS toxicity
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Inhibition of Inosine-5'-monophosphate Dehydrogenase from Bacillus anthracis: Mechanism Revealed by Pre-Steady-State Kinetics.
Biochemistry, 2016Co-Authors: Yang Wei, Petr Kuzmič, Gyan Modi, Lizbeth HedstromAbstract:Inosine-5'-monophosphate dehydrogenase (IMPDH) catalyzes the conversion of inosine 5'-monophosphate (IMP) to xanthosine 5'-monophosphate (XMP). The enzyme is an emerging target for antimicrobial therapy. The small molecule inhibitor A110 has been identified as a potent and selective inhibitor of IMPDHs from a variety of pathogenic microorganisms. A recent X-ray crystallographic study reported that the inhibitor binds to the NAD(+) cofactor site and forms a ternary complex with IMP. Here we report a pre-steady-state stopped-flow kinetic investigation of IMPDH from Bacillus anthracis designed to assess the kinetic significance of the crystallographic results. Stopped-flow kinetic experiments defined nine microscopic rate constants and two equilibrium constants that characterize both the catalytic cycle and details of the inhibition mechanism. In combination with steady-state initial rate studies, the results show that the inhibitor binds with high affinity (Kd ≈ 50 nM) predominantly to the covalent intermediate on the reaction pathway. Only a weak binding interaction (Kd ≈ 1 μM) is observed between the inhibitor and E·IMP. Thus, the E·IMP·A110 ternary complex, observed by X-ray crystallography, is largely kinetically irrelevant.
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Investigating the mechanism of disease in the RP10 form of retinitis pigmentosa.
Advances in experimental medicine and biology, 2009Co-Authors: Catherine J Spellicy, Lizbeth Hedstrom, Sara J Bowne, Lori S Sullivan, Garrett C. Cobb, Stephen P DaigerAbstract:Retinitis pigmentosa (RP) is a disease characterized by its vast heterogeneity. Many genes are associated with RP, and the disease causing mutations identified in these genes are even more numerous. To date there are 15 genes that cause autosomal dominant RP (adRP) alone. The role of some of these genes, while complex and not completely understood, is somewhat intuitive in that they are involved in pathways such as phototransduction. However, the role of other genes in retinal disease is not as predictable due to their ubiquitous function and/or expression. One such gene is inosine monophosphate dehydrogenase 1 (IMPDH1) IMPDH1 is a gene involved in de novo purine synthesis and is ubiquitously expressed. IMPDH1 mutations account for 2% of all adRP cases and are a rare cause of Leiber Congenital Amaurosis. Despite its ubiquitous expression missense mutations in this gene cause only retinal degeneration. This paradox of tissue specific disease in the presence of ubiquitous expression has only recently begun to be explained. We have shown in a recent study that novel retinal isoforms of IMPDH1 exist and may account for the tissue specificity of disease. We have gone on to characterize these retinal isoforms both in our laboratory and in collaboration with Dr. Lizbeth Hedstrom’s laboratory at Brandeis University (Waltham, MA) in order to understand more about them. We believe that through clarifying the mechanism of disease in RP10 we will be equipped to consider treatment options for this disease.
Stephen P Daiger - One of the best experts on this subject based on the ideXlab platform.
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Investigating the mechanism of disease in the RP10 form of retinitis pigmentosa.
Advances in experimental medicine and biology, 2009Co-Authors: Catherine J Spellicy, Lizbeth Hedstrom, Sara J Bowne, Lori S Sullivan, Garrett C. Cobb, Stephen P DaigerAbstract:Retinitis pigmentosa (RP) is a disease characterized by its vast heterogeneity. Many genes are associated with RP, and the disease causing mutations identified in these genes are even more numerous. To date there are 15 genes that cause autosomal dominant RP (adRP) alone. The role of some of these genes, while complex and not completely understood, is somewhat intuitive in that they are involved in pathways such as phototransduction. However, the role of other genes in retinal disease is not as predictable due to their ubiquitous function and/or expression. One such gene is inosine monophosphate dehydrogenase 1 (IMPDH1) IMPDH1 is a gene involved in de novo purine synthesis and is ubiquitously expressed. IMPDH1 mutations account for 2% of all adRP cases and are a rare cause of Leiber Congenital Amaurosis. Despite its ubiquitous expression missense mutations in this gene cause only retinal degeneration. This paradox of tissue specific disease in the presence of ubiquitous expression has only recently begun to be explained. We have shown in a recent study that novel retinal isoforms of IMPDH1 exist and may account for the tissue specificity of disease. We have gone on to characterize these retinal isoforms both in our laboratory and in collaboration with Dr. Lizbeth Hedstrom’s laboratory at Brandeis University (Waltham, MA) in order to understand more about them. We believe that through clarifying the mechanism of disease in RP10 we will be equipped to consider treatment options for this disease.
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IMP dehydrogenase type 1 associates with polyribosomes translating rhodopsin mRNA.
The Journal of biological chemistry, 2008Co-Authors: Sarah E. Mortimer, Sara J Bowne, Stephen P Daiger, Dharia A. Mcgrew, Nobuko Hamaguchi, Hoong Chuin Lim, Lizbeth HedstromAbstract:IMP dehydrogenase (IMPDH) catalyzes the pivotal step in guanine nucleotide biosynthesis. Here we show that both IMPDH type 1 (IMPDH1) and IMPDH type 2 are associated with polyribosomes, suggesting that these housekeeping proteins have an unanticipated role in translation regulation. This interaction is mediated by the subdomain, a region of disputed function that is the site of mutations that cause retinal degeneration. The retinal isoforms of IMPDH1 also associate with polyribosomes. The most common disease-causing mutation, D226N, disrupts the polyribosome association of at least one retinal IMPDH1 isoform. Finally, we find that IMPDH1 is associated with polyribosomes containing rhodopsin mRNA. Because any perturbation of rhodopsin expression can trigger apoptosis in photoreceptor cells, these observations suggest a likely pathological mechanism for IMPDH1-mediated hereditary blindness. We propose that IMPDH coordinates the translation of a set of mRNAs, perhaps by modulating localization or degradation.
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retinal isoforms of inosine 5 monophosphate dehydrogenase type 1 are poor nucleic acid binding proteins
Archives of Biochemistry and Biophysics, 2008Co-Authors: Garrett Cobb, Catherine J Spellicy, Sara J Bowne, Stephen P Daiger, Lizbeth HedstromAbstract:The RP 10 form of autosomal dominant retinitis pigmentosa (adRP) is caused by mutations in the widely expressed protein inosine 5′-monophosphate dehydrogenase type 1 (IMPDH1). These mutations have no effect on the enzymatic activity of IMPDH1, but do perturb the association of IMPDH1 with nucleic acids. Two newly discovered retinal-specific isoforms, IMPDH1(546) and IMPDH1(595), may provide the key to the photoreceptor specificity of disease (S.J. Bowne, Q. Liu, L.S. Sullivan, J. Zhu, C.J. Spellicy, C.B. Rickman, E.A. Pierce, S.P. Daiger, Invest. Ophthalmol. Vis. Sci. 47 (2006) 3754–3765). Here we express and characterize the normal IMPDH1(546) and IMPDH1(595), together with their adRP-linked variants, D226N. The enzymatic activity of the purified IMPDH1(546) and IMPDH1(595) and the D226N variants is indistinguishable from the canonical form. The intracellular distribution of IMPDH1(546) and IMPDH1(595) is also similar to the canonical IMPDH1 and unaffected by the D226N mutation. However, unlike the canonical IMPDH1, the retinal specific isoforms do not bind significant fractions of a random pool of oligonucleotides. This observation indicates that the C-terminal extension unique to the retinal isoforms blocks the nucleic acid binding site of the IMPDH1, and thus uniquely regulates protein function within photoreceptors.
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characterization of retinal inosine monophosphate dehydrogenase 1 in several mammalian species
Molecular Vision, 2007Co-Authors: Catherine J Spellicy, Qin Liu, Lori S Sullivan, Jingya Zhu, Eric A Pierce, Stephen P Daiger, Sara J BowneAbstract:PURPOSE The purpose of this study was to characterize the inosine monophosphate dehydrogenase 1 (IMPDH1) protein isoforms in mammalian retinas, in order to determine the species distribution of these variants and identify an optimal animal model for studying IMPDH1-associated retinal diseases. Mutations in IMPDH1 cause the RP10 form of autosomal dominant retinitis pigmentosa, and are a rare cause of Leber congenital amaurosis. METHODS Retinas from several mammalian species were obtained commercially. Human retinas were isolated by the San Diego Eye Bank and flash frozen within four hours post mortem. Proteins were isolated from retinal tissue using the PARIS protocol. Anti-IMPDH1 antibodies were used to visualize the IMDPH1 proteins on Western blots. RESULTS Transcript and protein analyses have shown that IMPDH1 undergoes alternate splicing to produce at least two retinal isoforms in both human and mouse. The relative abundance of these IMPDH1 isoforms is different between mouse and human. This study extends these findings by showing that the two IMPDH1 isoforms are also present in dog, rat, sheep, pig, and cow retina, but that, as with mouse, the relative abundances of these isoforms differ from those found in human retina. CONCLUSIONS The existence of two major retinal isoforms of the IMPDH1 protein is maintained across all mammalian species tested. The relative abundance of IMPDH1 proteins in human retina is unique in comparison to other mammalian species, indicating an apparent lack of an ideal model organism for human retinal IMPDH1 expression. Pig and/or sheep may prove to be potential model organisms based on the observed retinal isoform abundance in these species. These findings will aid future research in understanding the role of retinal-specific IMPDH1 proteins, and will contribute to research elucidating the pathophysiology associated with IMPDH1 missense mutations.
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why do mutations in the ubiquitously expressed housekeeping gene IMPDH1 cause retina specific photoreceptor degeneration
Investigative Ophthalmology & Visual Science, 2006Co-Authors: Sara J Bowne, Qin Liu, Lori S Sullivan, Jingya Zhu, Catherine J Spellicy, Catherine Bowes Rickman, Eric A Pierce, Stephen P DaigerAbstract:Retinitis pigmentosa (RP) is a heterogeneous form of inherited retinal degeneration that affects 100,000 individuals in the United States and approximately 1.5 million individuals worldwide.1 Initial symptoms of RP include night blindness followed by loss of peripheral vision. Vision loss progresses as a result of photoreceptor death from the midperipheral retina toward the macula, culminating in tunnel vision, legal blindness, and all too often, total loss of sight.2 RP is caused by mutations in many distinct genes. To date, 16 autosomal dominant, 16 autosomal recessive, and 6 X-linked forms have been identified in addition to many other syndromic, systemic, and complex forms (RetNet; http//:www.sph.uth.tmc.edu/RetNet/ provided in the public domain by the University of Texas Houston Health Science Center, Houston, TX). Several forms of RP are caused by mutations in photoreceptor-specific or abundant proteins involved in phototransduction or the visual cycle. Understanding the pathophysiology of these is well advanced due to the large amount already known about the pathways involved.3 Other forms of RP are caused by mutations in more widely expressed genes whose mechanisms of action are largely unknown. One such gene is inosine monophosphate dehydrogenase 1 (IMPDH1). Mutations in IMPDH1 cause the RP10 form of autosomal dominant RP (adRP) and are also a rare cause of isolated Leber congenital amaurosis (LCA).4-6 IMPDH1 is located on chromosome 7, region q32.1, and codes for the enzyme IMPDH type 1. Genes coding for IMPDH are found in all eukaryotes and most prokaryotes, and are highly conserved across species at both the gene and protein levels.7,8 Like most mammals, humans have an IMPDH1 and an IMPDH2 gene. These genes encode enzymes that are 84% identical at the amino acid level.9 All IMPDH proteins form active homotetramers that catalyze the rate-limiting step of de novo guanine synthesis by converting inosine monophosphate (IMP) to xanthosine monophosphate (XMP) with the reduction of NAD. Each IMPDH monomer is composed of an eight-stranded α/β barrel structure, which performs the enzymatic function, and a flanking subdomain, which is composed of two CBS regions similar to the cystathionine β-synthase gene. Human IMPDH1 and IMPDH2 enzymes have indistinguishable substrate affinities and catalytic activities, although they do show differences in inhibitor binding.10,11 Binding of single-stranded nucleic acids has recently been identified as a property of IMPDH proteins.12,13 In vitro and in vivo analysis demonstrates that several IMPDH species, including human IMPDH1 and IMPDH2, bind random pools of single-stranded nucleotides with nanomolar affinity.12 The biological role of the nucleic acid binding property of IMPDH is currently unknown.12 The expression of IMPDH in various tissues has been studied by several groups. Northern blot analyses show that IMPDH1 and IMPDH2 are expressed in most tissues, with the highest levels of IMPDH1 found in resting and activated peripheral blood lymphocytes. IMPDH2 levels are higher than IMPDH1 in all other tissues.7,14,15 IMPDH2 levels are even higher in cancerous cells, whereas IMPDH1 expression levels are not affected by transformation.16 Originally, three different IMPDH1 transcripts were identified and described in human cells and tissues.17 These transcripts differ in size (4.0, 2.7, and 2.5 kb) but contain identical coding sequences, derived from 14 exons, and identical 3′-untranslated regions (UTRs; for example, see Fig. 4A). The three transcripts differ only in splicing of three alternate untranslated 5′ exons, historically designated A, B, and C. Each of these transcripts encodes an identical protein that we refer to as “canonical IMPDH1,” a 55.6-kDa protein 514 amino acids in length. In contrast, the human IMPDH2 gene, though identical in exonic structure within the coding region, does not contain sequences homologous to the 5′ exons A, B, or C of IMPDH1, nor does it appear to have any additional exons preceding exon 1. Figure 4 Human IMPDH1 transcripts and proteins. (A) Genomic structure and three IMPDH1 transcripts originally reported by Gu et al.17 Data suggested that all three transcripts encoded the canonical IMPDH1 protein shown on the right. (B) Observed retinal transcripts ... Known IMPDH1 mutations cause retinal degeneration only, despite a much wider expression pattern.4,5,18 The disease mechanism of these mutations and the reason that they manifest as a retina-specific phenotype are currently unknown. One possibility is that IMPDH1 mutations are null and cause a loss of enzyme activity that only affects photoreceptors or that is compensated for by IMPDH2 in other tissues. However, several studies have shown that IMPDH1 mutations do not affect enzyme activity and hence make this an unlikely mechanism for disease.6,19,20 Another possibility is that the IMPDH1 mutations confer a gain of function or alteration of a function unique to photoreceptors. In support of this possibility, IMPDH1 mutations alter the affinity and/or specificity of the nucleic acid binding property of IMPDH1.6,20 How, or even if, this effect on nucleic acid binding is related to retinal disease is currently unknown. Although much is known about IMPDH1 expression and proteins in many human and mouse tissues, little is known about it in the retina. In this study, we used a variety of RNA and protein analyses to study retinal IMPDH1. Our expression analyses show that IMPDH1 is present at very high levels in the retina and that it is localized to the inner segment and synaptic terminals of photoreceptors. The predominant RNA and protein isoforms of IMPDH1 we identified in the retina are different from those that have been identified in other tissues. These unique isoforms are created via alternate splicing and/or the use of an in-frame, upstream initiation codon. Cross-species comparisons show that the unique protein regions of retinal IMPDH1 proteins are highly conserved, although our data from mice and humans indicate that the ratios of these unique isoforms vary between species. These experiments suggest that the pathophysiology of retinal degeneration is not caused by alteration of the canonical IMPDH1 protein, but rather acts through the unique retinal isoforms of IMPDH1.
Sara J Bowne - One of the best experts on this subject based on the ideXlab platform.
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Investigating the mechanism of disease in the RP10 form of retinitis pigmentosa.
Advances in experimental medicine and biology, 2009Co-Authors: Catherine J Spellicy, Lizbeth Hedstrom, Sara J Bowne, Lori S Sullivan, Garrett C. Cobb, Stephen P DaigerAbstract:Retinitis pigmentosa (RP) is a disease characterized by its vast heterogeneity. Many genes are associated with RP, and the disease causing mutations identified in these genes are even more numerous. To date there are 15 genes that cause autosomal dominant RP (adRP) alone. The role of some of these genes, while complex and not completely understood, is somewhat intuitive in that they are involved in pathways such as phototransduction. However, the role of other genes in retinal disease is not as predictable due to their ubiquitous function and/or expression. One such gene is inosine monophosphate dehydrogenase 1 (IMPDH1) IMPDH1 is a gene involved in de novo purine synthesis and is ubiquitously expressed. IMPDH1 mutations account for 2% of all adRP cases and are a rare cause of Leiber Congenital Amaurosis. Despite its ubiquitous expression missense mutations in this gene cause only retinal degeneration. This paradox of tissue specific disease in the presence of ubiquitous expression has only recently begun to be explained. We have shown in a recent study that novel retinal isoforms of IMPDH1 exist and may account for the tissue specificity of disease. We have gone on to characterize these retinal isoforms both in our laboratory and in collaboration with Dr. Lizbeth Hedstrom’s laboratory at Brandeis University (Waltham, MA) in order to understand more about them. We believe that through clarifying the mechanism of disease in RP10 we will be equipped to consider treatment options for this disease.
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IMP dehydrogenase type 1 associates with polyribosomes translating rhodopsin mRNA.
The Journal of biological chemistry, 2008Co-Authors: Sarah E. Mortimer, Sara J Bowne, Stephen P Daiger, Dharia A. Mcgrew, Nobuko Hamaguchi, Hoong Chuin Lim, Lizbeth HedstromAbstract:IMP dehydrogenase (IMPDH) catalyzes the pivotal step in guanine nucleotide biosynthesis. Here we show that both IMPDH type 1 (IMPDH1) and IMPDH type 2 are associated with polyribosomes, suggesting that these housekeeping proteins have an unanticipated role in translation regulation. This interaction is mediated by the subdomain, a region of disputed function that is the site of mutations that cause retinal degeneration. The retinal isoforms of IMPDH1 also associate with polyribosomes. The most common disease-causing mutation, D226N, disrupts the polyribosome association of at least one retinal IMPDH1 isoform. Finally, we find that IMPDH1 is associated with polyribosomes containing rhodopsin mRNA. Because any perturbation of rhodopsin expression can trigger apoptosis in photoreceptor cells, these observations suggest a likely pathological mechanism for IMPDH1-mediated hereditary blindness. We propose that IMPDH coordinates the translation of a set of mRNAs, perhaps by modulating localization or degradation.
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retinal isoforms of inosine 5 monophosphate dehydrogenase type 1 are poor nucleic acid binding proteins
Archives of Biochemistry and Biophysics, 2008Co-Authors: Garrett Cobb, Catherine J Spellicy, Sara J Bowne, Stephen P Daiger, Lizbeth HedstromAbstract:The RP 10 form of autosomal dominant retinitis pigmentosa (adRP) is caused by mutations in the widely expressed protein inosine 5′-monophosphate dehydrogenase type 1 (IMPDH1). These mutations have no effect on the enzymatic activity of IMPDH1, but do perturb the association of IMPDH1 with nucleic acids. Two newly discovered retinal-specific isoforms, IMPDH1(546) and IMPDH1(595), may provide the key to the photoreceptor specificity of disease (S.J. Bowne, Q. Liu, L.S. Sullivan, J. Zhu, C.J. Spellicy, C.B. Rickman, E.A. Pierce, S.P. Daiger, Invest. Ophthalmol. Vis. Sci. 47 (2006) 3754–3765). Here we express and characterize the normal IMPDH1(546) and IMPDH1(595), together with their adRP-linked variants, D226N. The enzymatic activity of the purified IMPDH1(546) and IMPDH1(595) and the D226N variants is indistinguishable from the canonical form. The intracellular distribution of IMPDH1(546) and IMPDH1(595) is also similar to the canonical IMPDH1 and unaffected by the D226N mutation. However, unlike the canonical IMPDH1, the retinal specific isoforms do not bind significant fractions of a random pool of oligonucleotides. This observation indicates that the C-terminal extension unique to the retinal isoforms blocks the nucleic acid binding site of the IMPDH1, and thus uniquely regulates protein function within photoreceptors.
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characterization of retinal inosine monophosphate dehydrogenase 1 in several mammalian species
Molecular Vision, 2007Co-Authors: Catherine J Spellicy, Qin Liu, Lori S Sullivan, Jingya Zhu, Eric A Pierce, Stephen P Daiger, Sara J BowneAbstract:PURPOSE The purpose of this study was to characterize the inosine monophosphate dehydrogenase 1 (IMPDH1) protein isoforms in mammalian retinas, in order to determine the species distribution of these variants and identify an optimal animal model for studying IMPDH1-associated retinal diseases. Mutations in IMPDH1 cause the RP10 form of autosomal dominant retinitis pigmentosa, and are a rare cause of Leber congenital amaurosis. METHODS Retinas from several mammalian species were obtained commercially. Human retinas were isolated by the San Diego Eye Bank and flash frozen within four hours post mortem. Proteins were isolated from retinal tissue using the PARIS protocol. Anti-IMPDH1 antibodies were used to visualize the IMDPH1 proteins on Western blots. RESULTS Transcript and protein analyses have shown that IMPDH1 undergoes alternate splicing to produce at least two retinal isoforms in both human and mouse. The relative abundance of these IMPDH1 isoforms is different between mouse and human. This study extends these findings by showing that the two IMPDH1 isoforms are also present in dog, rat, sheep, pig, and cow retina, but that, as with mouse, the relative abundances of these isoforms differ from those found in human retina. CONCLUSIONS The existence of two major retinal isoforms of the IMPDH1 protein is maintained across all mammalian species tested. The relative abundance of IMPDH1 proteins in human retina is unique in comparison to other mammalian species, indicating an apparent lack of an ideal model organism for human retinal IMPDH1 expression. Pig and/or sheep may prove to be potential model organisms based on the observed retinal isoform abundance in these species. These findings will aid future research in understanding the role of retinal-specific IMPDH1 proteins, and will contribute to research elucidating the pathophysiology associated with IMPDH1 missense mutations.
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why do mutations in the ubiquitously expressed housekeeping gene IMPDH1 cause retina specific photoreceptor degeneration
Investigative Ophthalmology & Visual Science, 2006Co-Authors: Sara J Bowne, Qin Liu, Lori S Sullivan, Jingya Zhu, Catherine J Spellicy, Catherine Bowes Rickman, Eric A Pierce, Stephen P DaigerAbstract:Retinitis pigmentosa (RP) is a heterogeneous form of inherited retinal degeneration that affects 100,000 individuals in the United States and approximately 1.5 million individuals worldwide.1 Initial symptoms of RP include night blindness followed by loss of peripheral vision. Vision loss progresses as a result of photoreceptor death from the midperipheral retina toward the macula, culminating in tunnel vision, legal blindness, and all too often, total loss of sight.2 RP is caused by mutations in many distinct genes. To date, 16 autosomal dominant, 16 autosomal recessive, and 6 X-linked forms have been identified in addition to many other syndromic, systemic, and complex forms (RetNet; http//:www.sph.uth.tmc.edu/RetNet/ provided in the public domain by the University of Texas Houston Health Science Center, Houston, TX). Several forms of RP are caused by mutations in photoreceptor-specific or abundant proteins involved in phototransduction or the visual cycle. Understanding the pathophysiology of these is well advanced due to the large amount already known about the pathways involved.3 Other forms of RP are caused by mutations in more widely expressed genes whose mechanisms of action are largely unknown. One such gene is inosine monophosphate dehydrogenase 1 (IMPDH1). Mutations in IMPDH1 cause the RP10 form of autosomal dominant RP (adRP) and are also a rare cause of isolated Leber congenital amaurosis (LCA).4-6 IMPDH1 is located on chromosome 7, region q32.1, and codes for the enzyme IMPDH type 1. Genes coding for IMPDH are found in all eukaryotes and most prokaryotes, and are highly conserved across species at both the gene and protein levels.7,8 Like most mammals, humans have an IMPDH1 and an IMPDH2 gene. These genes encode enzymes that are 84% identical at the amino acid level.9 All IMPDH proteins form active homotetramers that catalyze the rate-limiting step of de novo guanine synthesis by converting inosine monophosphate (IMP) to xanthosine monophosphate (XMP) with the reduction of NAD. Each IMPDH monomer is composed of an eight-stranded α/β barrel structure, which performs the enzymatic function, and a flanking subdomain, which is composed of two CBS regions similar to the cystathionine β-synthase gene. Human IMPDH1 and IMPDH2 enzymes have indistinguishable substrate affinities and catalytic activities, although they do show differences in inhibitor binding.10,11 Binding of single-stranded nucleic acids has recently been identified as a property of IMPDH proteins.12,13 In vitro and in vivo analysis demonstrates that several IMPDH species, including human IMPDH1 and IMPDH2, bind random pools of single-stranded nucleotides with nanomolar affinity.12 The biological role of the nucleic acid binding property of IMPDH is currently unknown.12 The expression of IMPDH in various tissues has been studied by several groups. Northern blot analyses show that IMPDH1 and IMPDH2 are expressed in most tissues, with the highest levels of IMPDH1 found in resting and activated peripheral blood lymphocytes. IMPDH2 levels are higher than IMPDH1 in all other tissues.7,14,15 IMPDH2 levels are even higher in cancerous cells, whereas IMPDH1 expression levels are not affected by transformation.16 Originally, three different IMPDH1 transcripts were identified and described in human cells and tissues.17 These transcripts differ in size (4.0, 2.7, and 2.5 kb) but contain identical coding sequences, derived from 14 exons, and identical 3′-untranslated regions (UTRs; for example, see Fig. 4A). The three transcripts differ only in splicing of three alternate untranslated 5′ exons, historically designated A, B, and C. Each of these transcripts encodes an identical protein that we refer to as “canonical IMPDH1,” a 55.6-kDa protein 514 amino acids in length. In contrast, the human IMPDH2 gene, though identical in exonic structure within the coding region, does not contain sequences homologous to the 5′ exons A, B, or C of IMPDH1, nor does it appear to have any additional exons preceding exon 1. Figure 4 Human IMPDH1 transcripts and proteins. (A) Genomic structure and three IMPDH1 transcripts originally reported by Gu et al.17 Data suggested that all three transcripts encoded the canonical IMPDH1 protein shown on the right. (B) Observed retinal transcripts ... Known IMPDH1 mutations cause retinal degeneration only, despite a much wider expression pattern.4,5,18 The disease mechanism of these mutations and the reason that they manifest as a retina-specific phenotype are currently unknown. One possibility is that IMPDH1 mutations are null and cause a loss of enzyme activity that only affects photoreceptors or that is compensated for by IMPDH2 in other tissues. However, several studies have shown that IMPDH1 mutations do not affect enzyme activity and hence make this an unlikely mechanism for disease.6,19,20 Another possibility is that the IMPDH1 mutations confer a gain of function or alteration of a function unique to photoreceptors. In support of this possibility, IMPDH1 mutations alter the affinity and/or specificity of the nucleic acid binding property of IMPDH1.6,20 How, or even if, this effect on nucleic acid binding is related to retinal disease is currently unknown. Although much is known about IMPDH1 expression and proteins in many human and mouse tissues, little is known about it in the retina. In this study, we used a variety of RNA and protein analyses to study retinal IMPDH1. Our expression analyses show that IMPDH1 is present at very high levels in the retina and that it is localized to the inner segment and synaptic terminals of photoreceptors. The predominant RNA and protein isoforms of IMPDH1 we identified in the retina are different from those that have been identified in other tissues. These unique isoforms are created via alternate splicing and/or the use of an in-frame, upstream initiation codon. Cross-species comparisons show that the unique protein regions of retinal IMPDH1 proteins are highly conserved, although our data from mice and humans indicate that the ratios of these unique isoforms vary between species. These experiments suggest that the pathophysiology of retinal degeneration is not caused by alteration of the canonical IMPDH1 protein, but rather acts through the unique retinal isoforms of IMPDH1.
Catherine J Spellicy - One of the best experts on this subject based on the ideXlab platform.
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Investigating the mechanism of disease in the RP10 form of retinitis pigmentosa.
Advances in experimental medicine and biology, 2009Co-Authors: Catherine J Spellicy, Lizbeth Hedstrom, Sara J Bowne, Lori S Sullivan, Garrett C. Cobb, Stephen P DaigerAbstract:Retinitis pigmentosa (RP) is a disease characterized by its vast heterogeneity. Many genes are associated with RP, and the disease causing mutations identified in these genes are even more numerous. To date there are 15 genes that cause autosomal dominant RP (adRP) alone. The role of some of these genes, while complex and not completely understood, is somewhat intuitive in that they are involved in pathways such as phototransduction. However, the role of other genes in retinal disease is not as predictable due to their ubiquitous function and/or expression. One such gene is inosine monophosphate dehydrogenase 1 (IMPDH1) IMPDH1 is a gene involved in de novo purine synthesis and is ubiquitously expressed. IMPDH1 mutations account for 2% of all adRP cases and are a rare cause of Leiber Congenital Amaurosis. Despite its ubiquitous expression missense mutations in this gene cause only retinal degeneration. This paradox of tissue specific disease in the presence of ubiquitous expression has only recently begun to be explained. We have shown in a recent study that novel retinal isoforms of IMPDH1 exist and may account for the tissue specificity of disease. We have gone on to characterize these retinal isoforms both in our laboratory and in collaboration with Dr. Lizbeth Hedstrom’s laboratory at Brandeis University (Waltham, MA) in order to understand more about them. We believe that through clarifying the mechanism of disease in RP10 we will be equipped to consider treatment options for this disease.
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retinal isoforms of inosine 5 monophosphate dehydrogenase type 1 are poor nucleic acid binding proteins
Archives of Biochemistry and Biophysics, 2008Co-Authors: Garrett Cobb, Catherine J Spellicy, Sara J Bowne, Stephen P Daiger, Lizbeth HedstromAbstract:The RP 10 form of autosomal dominant retinitis pigmentosa (adRP) is caused by mutations in the widely expressed protein inosine 5′-monophosphate dehydrogenase type 1 (IMPDH1). These mutations have no effect on the enzymatic activity of IMPDH1, but do perturb the association of IMPDH1 with nucleic acids. Two newly discovered retinal-specific isoforms, IMPDH1(546) and IMPDH1(595), may provide the key to the photoreceptor specificity of disease (S.J. Bowne, Q. Liu, L.S. Sullivan, J. Zhu, C.J. Spellicy, C.B. Rickman, E.A. Pierce, S.P. Daiger, Invest. Ophthalmol. Vis. Sci. 47 (2006) 3754–3765). Here we express and characterize the normal IMPDH1(546) and IMPDH1(595), together with their adRP-linked variants, D226N. The enzymatic activity of the purified IMPDH1(546) and IMPDH1(595) and the D226N variants is indistinguishable from the canonical form. The intracellular distribution of IMPDH1(546) and IMPDH1(595) is also similar to the canonical IMPDH1 and unaffected by the D226N mutation. However, unlike the canonical IMPDH1, the retinal specific isoforms do not bind significant fractions of a random pool of oligonucleotides. This observation indicates that the C-terminal extension unique to the retinal isoforms blocks the nucleic acid binding site of the IMPDH1, and thus uniquely regulates protein function within photoreceptors.
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characterization of retinal inosine monophosphate dehydrogenase 1 in several mammalian species
Molecular Vision, 2007Co-Authors: Catherine J Spellicy, Qin Liu, Lori S Sullivan, Jingya Zhu, Eric A Pierce, Stephen P Daiger, Sara J BowneAbstract:PURPOSE The purpose of this study was to characterize the inosine monophosphate dehydrogenase 1 (IMPDH1) protein isoforms in mammalian retinas, in order to determine the species distribution of these variants and identify an optimal animal model for studying IMPDH1-associated retinal diseases. Mutations in IMPDH1 cause the RP10 form of autosomal dominant retinitis pigmentosa, and are a rare cause of Leber congenital amaurosis. METHODS Retinas from several mammalian species were obtained commercially. Human retinas were isolated by the San Diego Eye Bank and flash frozen within four hours post mortem. Proteins were isolated from retinal tissue using the PARIS protocol. Anti-IMPDH1 antibodies were used to visualize the IMDPH1 proteins on Western blots. RESULTS Transcript and protein analyses have shown that IMPDH1 undergoes alternate splicing to produce at least two retinal isoforms in both human and mouse. The relative abundance of these IMPDH1 isoforms is different between mouse and human. This study extends these findings by showing that the two IMPDH1 isoforms are also present in dog, rat, sheep, pig, and cow retina, but that, as with mouse, the relative abundances of these isoforms differ from those found in human retina. CONCLUSIONS The existence of two major retinal isoforms of the IMPDH1 protein is maintained across all mammalian species tested. The relative abundance of IMPDH1 proteins in human retina is unique in comparison to other mammalian species, indicating an apparent lack of an ideal model organism for human retinal IMPDH1 expression. Pig and/or sheep may prove to be potential model organisms based on the observed retinal isoform abundance in these species. These findings will aid future research in understanding the role of retinal-specific IMPDH1 proteins, and will contribute to research elucidating the pathophysiology associated with IMPDH1 missense mutations.
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why do mutations in the ubiquitously expressed housekeeping gene IMPDH1 cause retina specific photoreceptor degeneration
Investigative Ophthalmology & Visual Science, 2006Co-Authors: Sara J Bowne, Qin Liu, Lori S Sullivan, Jingya Zhu, Catherine J Spellicy, Catherine Bowes Rickman, Eric A Pierce, Stephen P DaigerAbstract:Retinitis pigmentosa (RP) is a heterogeneous form of inherited retinal degeneration that affects 100,000 individuals in the United States and approximately 1.5 million individuals worldwide.1 Initial symptoms of RP include night blindness followed by loss of peripheral vision. Vision loss progresses as a result of photoreceptor death from the midperipheral retina toward the macula, culminating in tunnel vision, legal blindness, and all too often, total loss of sight.2 RP is caused by mutations in many distinct genes. To date, 16 autosomal dominant, 16 autosomal recessive, and 6 X-linked forms have been identified in addition to many other syndromic, systemic, and complex forms (RetNet; http//:www.sph.uth.tmc.edu/RetNet/ provided in the public domain by the University of Texas Houston Health Science Center, Houston, TX). Several forms of RP are caused by mutations in photoreceptor-specific or abundant proteins involved in phototransduction or the visual cycle. Understanding the pathophysiology of these is well advanced due to the large amount already known about the pathways involved.3 Other forms of RP are caused by mutations in more widely expressed genes whose mechanisms of action are largely unknown. One such gene is inosine monophosphate dehydrogenase 1 (IMPDH1). Mutations in IMPDH1 cause the RP10 form of autosomal dominant RP (adRP) and are also a rare cause of isolated Leber congenital amaurosis (LCA).4-6 IMPDH1 is located on chromosome 7, region q32.1, and codes for the enzyme IMPDH type 1. Genes coding for IMPDH are found in all eukaryotes and most prokaryotes, and are highly conserved across species at both the gene and protein levels.7,8 Like most mammals, humans have an IMPDH1 and an IMPDH2 gene. These genes encode enzymes that are 84% identical at the amino acid level.9 All IMPDH proteins form active homotetramers that catalyze the rate-limiting step of de novo guanine synthesis by converting inosine monophosphate (IMP) to xanthosine monophosphate (XMP) with the reduction of NAD. Each IMPDH monomer is composed of an eight-stranded α/β barrel structure, which performs the enzymatic function, and a flanking subdomain, which is composed of two CBS regions similar to the cystathionine β-synthase gene. Human IMPDH1 and IMPDH2 enzymes have indistinguishable substrate affinities and catalytic activities, although they do show differences in inhibitor binding.10,11 Binding of single-stranded nucleic acids has recently been identified as a property of IMPDH proteins.12,13 In vitro and in vivo analysis demonstrates that several IMPDH species, including human IMPDH1 and IMPDH2, bind random pools of single-stranded nucleotides with nanomolar affinity.12 The biological role of the nucleic acid binding property of IMPDH is currently unknown.12 The expression of IMPDH in various tissues has been studied by several groups. Northern blot analyses show that IMPDH1 and IMPDH2 are expressed in most tissues, with the highest levels of IMPDH1 found in resting and activated peripheral blood lymphocytes. IMPDH2 levels are higher than IMPDH1 in all other tissues.7,14,15 IMPDH2 levels are even higher in cancerous cells, whereas IMPDH1 expression levels are not affected by transformation.16 Originally, three different IMPDH1 transcripts were identified and described in human cells and tissues.17 These transcripts differ in size (4.0, 2.7, and 2.5 kb) but contain identical coding sequences, derived from 14 exons, and identical 3′-untranslated regions (UTRs; for example, see Fig. 4A). The three transcripts differ only in splicing of three alternate untranslated 5′ exons, historically designated A, B, and C. Each of these transcripts encodes an identical protein that we refer to as “canonical IMPDH1,” a 55.6-kDa protein 514 amino acids in length. In contrast, the human IMPDH2 gene, though identical in exonic structure within the coding region, does not contain sequences homologous to the 5′ exons A, B, or C of IMPDH1, nor does it appear to have any additional exons preceding exon 1. Figure 4 Human IMPDH1 transcripts and proteins. (A) Genomic structure and three IMPDH1 transcripts originally reported by Gu et al.17 Data suggested that all three transcripts encoded the canonical IMPDH1 protein shown on the right. (B) Observed retinal transcripts ... Known IMPDH1 mutations cause retinal degeneration only, despite a much wider expression pattern.4,5,18 The disease mechanism of these mutations and the reason that they manifest as a retina-specific phenotype are currently unknown. One possibility is that IMPDH1 mutations are null and cause a loss of enzyme activity that only affects photoreceptors or that is compensated for by IMPDH2 in other tissues. However, several studies have shown that IMPDH1 mutations do not affect enzyme activity and hence make this an unlikely mechanism for disease.6,19,20 Another possibility is that the IMPDH1 mutations confer a gain of function or alteration of a function unique to photoreceptors. In support of this possibility, IMPDH1 mutations alter the affinity and/or specificity of the nucleic acid binding property of IMPDH1.6,20 How, or even if, this effect on nucleic acid binding is related to retinal disease is currently unknown. Although much is known about IMPDH1 expression and proteins in many human and mouse tissues, little is known about it in the retina. In this study, we used a variety of RNA and protein analyses to study retinal IMPDH1. Our expression analyses show that IMPDH1 is present at very high levels in the retina and that it is localized to the inner segment and synaptic terminals of photoreceptors. The predominant RNA and protein isoforms of IMPDH1 we identified in the retina are different from those that have been identified in other tissues. These unique isoforms are created via alternate splicing and/or the use of an in-frame, upstream initiation codon. Cross-species comparisons show that the unique protein regions of retinal IMPDH1 proteins are highly conserved, although our data from mice and humans indicate that the ratios of these unique isoforms vary between species. These experiments suggest that the pathophysiology of retinal degeneration is not caused by alteration of the canonical IMPDH1 protein, but rather acts through the unique retinal isoforms of IMPDH1.
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Spectrum and frequency of mutations in IMPDH1 associated with autosomal dominant retinitis pigmentosa and leber congenital amaurosis.
Investigative ophthalmology & visual science, 2006Co-Authors: Sara J Bowne, Lizbeth Hedstrom, Lori S Sullivan, Jingya Zhu, Catherine J Spellicy, Sarah E. Mortimer, Anisa I. Gire, Dianna K. Hughbanks-wheaton, David G. Birch, Richard A. LewisAbstract:Retinitis pigmentosa (RP) is a progressive form of retinal degeneration that affects approximately 1.5 million individuals worldwide.1 Genes and mutations causing RP are exceptionally heterogeneous. To date, 14 autosomal dominant, 15 autosomal recessive, and 5 X-linked forms of RP have been identified, in addition to many other syndromic, systemic, and complex forms (RetNet, http://www.sph.uth.tmc.edu/Ret-Net). Further, many distinct pathogenic mutations have been identified in each RP gene, and different mutations in the same gene may cause distinctly different forms of retinal disease. Determining the types of mutations and the range of phenotypes associated with each RP gene is one of the first steps to understanding the pathophysiologic mechanisms that lead to photoreceptor death, a crucial step in the development of treatments. One RP gene that has not been examined in a wide range of patients with inherited retinal disease but is likely to contribute to distinct phenotypes, is inosine monophosphate dehydrogenase type I (IMPDH1). Mutations in IMPDH1 cause the RP10 form of autosomal dominant RP (adRP).2,3 IMPDH1 is located on chromosome 7q32.1 and encodes the enzyme IMPDH1. IMPDH proteins form homotetramers and catalyze the ratelimiting step of de novo guanine synthesis by oxidizing IMP to xanthosine-5′-monophosphate (XMP) with reduction of nicotinamide adenine dinucleotide (NAD). IMPDH genes are found in virtually every organism, and the gene and amino acid sequences are highly conserved across species. Humans have two IMPDH genes, IMPDH1 and IMPDH2, both expressed in a wide range of tissues.3-6 Two IMPDH1 mutations, Arg224Pro and Asp226Asn, were identified by linkage mapping and positional cloning in three adRP families.2,3 A reasonable prediction is that these missense mutations cause photoreceptor degeneration because of reduced enzyme activity and subsequent reduction in guanine nucleotide concentration. However, contrary to expectations, two studies demonstrate that these mutations do not affect enzyme activity or tetramer formation.7,8 Thus, an important unanswered question is the nature of the functional consequences of IMPDH1 mutations that lead to retinal degeneration. Recent research shows that IMPDH binds single-stranded nucleic acids, suggesting another possible mechanism by which IMPDH1 mutations could cause retinal disease.9 In vitro and in vivo assays demonstrate that several IMPDH species, including human types I and II, bind random pools of single-stranded nucleotides via a protein subdomain composed of two CBS domains, named for homologous domains in cystathionine β-synthase (CBS). IMPDH can be found in both the cytoplasm and the nucleus of cultured cells; it binds both RNA and single-stranded DNA but does not bind to cognate RNA.9 Additional studies show that the Arg224Pro and Asp226Asn adRP mutations affect the affinity and the specificity of IMPDH1 nucleic acid binding and suggest that testing this functional property will assist in determining pathogenicity of novel IMPDH1 variants.8 In this study, we surveyed a large population of patients with adRP to determine the range and the frequency of IMPDH1 mutations. The clinical heterogeneity of mutations in genes associated with retinal degeneration has been demonstrated many times.10-13 Therefore, we also analyzed patients with a variety of other inherited retinal degenerations, specifically autosomal recessive RP (arRP), macular degeneration (MD), and Leber congenital amaurosis (LCA), to investigate the possibility that mutations in IMPDH1 cause alternate phenotypes. The enzymatic activity and nucleic acid binding properties of each novel IMPDH1 protein variant identified in these patients were also determined and used to infer pathogenicity.
Lori S Sullivan - One of the best experts on this subject based on the ideXlab platform.
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Investigating the mechanism of disease in the RP10 form of retinitis pigmentosa.
Advances in experimental medicine and biology, 2009Co-Authors: Catherine J Spellicy, Lizbeth Hedstrom, Sara J Bowne, Lori S Sullivan, Garrett C. Cobb, Stephen P DaigerAbstract:Retinitis pigmentosa (RP) is a disease characterized by its vast heterogeneity. Many genes are associated with RP, and the disease causing mutations identified in these genes are even more numerous. To date there are 15 genes that cause autosomal dominant RP (adRP) alone. The role of some of these genes, while complex and not completely understood, is somewhat intuitive in that they are involved in pathways such as phototransduction. However, the role of other genes in retinal disease is not as predictable due to their ubiquitous function and/or expression. One such gene is inosine monophosphate dehydrogenase 1 (IMPDH1) IMPDH1 is a gene involved in de novo purine synthesis and is ubiquitously expressed. IMPDH1 mutations account for 2% of all adRP cases and are a rare cause of Leiber Congenital Amaurosis. Despite its ubiquitous expression missense mutations in this gene cause only retinal degeneration. This paradox of tissue specific disease in the presence of ubiquitous expression has only recently begun to be explained. We have shown in a recent study that novel retinal isoforms of IMPDH1 exist and may account for the tissue specificity of disease. We have gone on to characterize these retinal isoforms both in our laboratory and in collaboration with Dr. Lizbeth Hedstrom’s laboratory at Brandeis University (Waltham, MA) in order to understand more about them. We believe that through clarifying the mechanism of disease in RP10 we will be equipped to consider treatment options for this disease.
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characterization of retinal inosine monophosphate dehydrogenase 1 in several mammalian species
Molecular Vision, 2007Co-Authors: Catherine J Spellicy, Qin Liu, Lori S Sullivan, Jingya Zhu, Eric A Pierce, Stephen P Daiger, Sara J BowneAbstract:PURPOSE The purpose of this study was to characterize the inosine monophosphate dehydrogenase 1 (IMPDH1) protein isoforms in mammalian retinas, in order to determine the species distribution of these variants and identify an optimal animal model for studying IMPDH1-associated retinal diseases. Mutations in IMPDH1 cause the RP10 form of autosomal dominant retinitis pigmentosa, and are a rare cause of Leber congenital amaurosis. METHODS Retinas from several mammalian species were obtained commercially. Human retinas were isolated by the San Diego Eye Bank and flash frozen within four hours post mortem. Proteins were isolated from retinal tissue using the PARIS protocol. Anti-IMPDH1 antibodies were used to visualize the IMDPH1 proteins on Western blots. RESULTS Transcript and protein analyses have shown that IMPDH1 undergoes alternate splicing to produce at least two retinal isoforms in both human and mouse. The relative abundance of these IMPDH1 isoforms is different between mouse and human. This study extends these findings by showing that the two IMPDH1 isoforms are also present in dog, rat, sheep, pig, and cow retina, but that, as with mouse, the relative abundances of these isoforms differ from those found in human retina. CONCLUSIONS The existence of two major retinal isoforms of the IMPDH1 protein is maintained across all mammalian species tested. The relative abundance of IMPDH1 proteins in human retina is unique in comparison to other mammalian species, indicating an apparent lack of an ideal model organism for human retinal IMPDH1 expression. Pig and/or sheep may prove to be potential model organisms based on the observed retinal isoform abundance in these species. These findings will aid future research in understanding the role of retinal-specific IMPDH1 proteins, and will contribute to research elucidating the pathophysiology associated with IMPDH1 missense mutations.
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why do mutations in the ubiquitously expressed housekeeping gene IMPDH1 cause retina specific photoreceptor degeneration
Investigative Ophthalmology & Visual Science, 2006Co-Authors: Sara J Bowne, Qin Liu, Lori S Sullivan, Jingya Zhu, Catherine J Spellicy, Catherine Bowes Rickman, Eric A Pierce, Stephen P DaigerAbstract:Retinitis pigmentosa (RP) is a heterogeneous form of inherited retinal degeneration that affects 100,000 individuals in the United States and approximately 1.5 million individuals worldwide.1 Initial symptoms of RP include night blindness followed by loss of peripheral vision. Vision loss progresses as a result of photoreceptor death from the midperipheral retina toward the macula, culminating in tunnel vision, legal blindness, and all too often, total loss of sight.2 RP is caused by mutations in many distinct genes. To date, 16 autosomal dominant, 16 autosomal recessive, and 6 X-linked forms have been identified in addition to many other syndromic, systemic, and complex forms (RetNet; http//:www.sph.uth.tmc.edu/RetNet/ provided in the public domain by the University of Texas Houston Health Science Center, Houston, TX). Several forms of RP are caused by mutations in photoreceptor-specific or abundant proteins involved in phototransduction or the visual cycle. Understanding the pathophysiology of these is well advanced due to the large amount already known about the pathways involved.3 Other forms of RP are caused by mutations in more widely expressed genes whose mechanisms of action are largely unknown. One such gene is inosine monophosphate dehydrogenase 1 (IMPDH1). Mutations in IMPDH1 cause the RP10 form of autosomal dominant RP (adRP) and are also a rare cause of isolated Leber congenital amaurosis (LCA).4-6 IMPDH1 is located on chromosome 7, region q32.1, and codes for the enzyme IMPDH type 1. Genes coding for IMPDH are found in all eukaryotes and most prokaryotes, and are highly conserved across species at both the gene and protein levels.7,8 Like most mammals, humans have an IMPDH1 and an IMPDH2 gene. These genes encode enzymes that are 84% identical at the amino acid level.9 All IMPDH proteins form active homotetramers that catalyze the rate-limiting step of de novo guanine synthesis by converting inosine monophosphate (IMP) to xanthosine monophosphate (XMP) with the reduction of NAD. Each IMPDH monomer is composed of an eight-stranded α/β barrel structure, which performs the enzymatic function, and a flanking subdomain, which is composed of two CBS regions similar to the cystathionine β-synthase gene. Human IMPDH1 and IMPDH2 enzymes have indistinguishable substrate affinities and catalytic activities, although they do show differences in inhibitor binding.10,11 Binding of single-stranded nucleic acids has recently been identified as a property of IMPDH proteins.12,13 In vitro and in vivo analysis demonstrates that several IMPDH species, including human IMPDH1 and IMPDH2, bind random pools of single-stranded nucleotides with nanomolar affinity.12 The biological role of the nucleic acid binding property of IMPDH is currently unknown.12 The expression of IMPDH in various tissues has been studied by several groups. Northern blot analyses show that IMPDH1 and IMPDH2 are expressed in most tissues, with the highest levels of IMPDH1 found in resting and activated peripheral blood lymphocytes. IMPDH2 levels are higher than IMPDH1 in all other tissues.7,14,15 IMPDH2 levels are even higher in cancerous cells, whereas IMPDH1 expression levels are not affected by transformation.16 Originally, three different IMPDH1 transcripts were identified and described in human cells and tissues.17 These transcripts differ in size (4.0, 2.7, and 2.5 kb) but contain identical coding sequences, derived from 14 exons, and identical 3′-untranslated regions (UTRs; for example, see Fig. 4A). The three transcripts differ only in splicing of three alternate untranslated 5′ exons, historically designated A, B, and C. Each of these transcripts encodes an identical protein that we refer to as “canonical IMPDH1,” a 55.6-kDa protein 514 amino acids in length. In contrast, the human IMPDH2 gene, though identical in exonic structure within the coding region, does not contain sequences homologous to the 5′ exons A, B, or C of IMPDH1, nor does it appear to have any additional exons preceding exon 1. Figure 4 Human IMPDH1 transcripts and proteins. (A) Genomic structure and three IMPDH1 transcripts originally reported by Gu et al.17 Data suggested that all three transcripts encoded the canonical IMPDH1 protein shown on the right. (B) Observed retinal transcripts ... Known IMPDH1 mutations cause retinal degeneration only, despite a much wider expression pattern.4,5,18 The disease mechanism of these mutations and the reason that they manifest as a retina-specific phenotype are currently unknown. One possibility is that IMPDH1 mutations are null and cause a loss of enzyme activity that only affects photoreceptors or that is compensated for by IMPDH2 in other tissues. However, several studies have shown that IMPDH1 mutations do not affect enzyme activity and hence make this an unlikely mechanism for disease.6,19,20 Another possibility is that the IMPDH1 mutations confer a gain of function or alteration of a function unique to photoreceptors. In support of this possibility, IMPDH1 mutations alter the affinity and/or specificity of the nucleic acid binding property of IMPDH1.6,20 How, or even if, this effect on nucleic acid binding is related to retinal disease is currently unknown. Although much is known about IMPDH1 expression and proteins in many human and mouse tissues, little is known about it in the retina. In this study, we used a variety of RNA and protein analyses to study retinal IMPDH1. Our expression analyses show that IMPDH1 is present at very high levels in the retina and that it is localized to the inner segment and synaptic terminals of photoreceptors. The predominant RNA and protein isoforms of IMPDH1 we identified in the retina are different from those that have been identified in other tissues. These unique isoforms are created via alternate splicing and/or the use of an in-frame, upstream initiation codon. Cross-species comparisons show that the unique protein regions of retinal IMPDH1 proteins are highly conserved, although our data from mice and humans indicate that the ratios of these unique isoforms vary between species. These experiments suggest that the pathophysiology of retinal degeneration is not caused by alteration of the canonical IMPDH1 protein, but rather acts through the unique retinal isoforms of IMPDH1.
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Spectrum and frequency of mutations in IMPDH1 associated with autosomal dominant retinitis pigmentosa and leber congenital amaurosis.
Investigative ophthalmology & visual science, 2006Co-Authors: Sara J Bowne, Lizbeth Hedstrom, Lori S Sullivan, Jingya Zhu, Catherine J Spellicy, Sarah E. Mortimer, Anisa I. Gire, Dianna K. Hughbanks-wheaton, David G. Birch, Richard A. LewisAbstract:Retinitis pigmentosa (RP) is a progressive form of retinal degeneration that affects approximately 1.5 million individuals worldwide.1 Genes and mutations causing RP are exceptionally heterogeneous. To date, 14 autosomal dominant, 15 autosomal recessive, and 5 X-linked forms of RP have been identified, in addition to many other syndromic, systemic, and complex forms (RetNet, http://www.sph.uth.tmc.edu/Ret-Net). Further, many distinct pathogenic mutations have been identified in each RP gene, and different mutations in the same gene may cause distinctly different forms of retinal disease. Determining the types of mutations and the range of phenotypes associated with each RP gene is one of the first steps to understanding the pathophysiologic mechanisms that lead to photoreceptor death, a crucial step in the development of treatments. One RP gene that has not been examined in a wide range of patients with inherited retinal disease but is likely to contribute to distinct phenotypes, is inosine monophosphate dehydrogenase type I (IMPDH1). Mutations in IMPDH1 cause the RP10 form of autosomal dominant RP (adRP).2,3 IMPDH1 is located on chromosome 7q32.1 and encodes the enzyme IMPDH1. IMPDH proteins form homotetramers and catalyze the ratelimiting step of de novo guanine synthesis by oxidizing IMP to xanthosine-5′-monophosphate (XMP) with reduction of nicotinamide adenine dinucleotide (NAD). IMPDH genes are found in virtually every organism, and the gene and amino acid sequences are highly conserved across species. Humans have two IMPDH genes, IMPDH1 and IMPDH2, both expressed in a wide range of tissues.3-6 Two IMPDH1 mutations, Arg224Pro and Asp226Asn, were identified by linkage mapping and positional cloning in three adRP families.2,3 A reasonable prediction is that these missense mutations cause photoreceptor degeneration because of reduced enzyme activity and subsequent reduction in guanine nucleotide concentration. However, contrary to expectations, two studies demonstrate that these mutations do not affect enzyme activity or tetramer formation.7,8 Thus, an important unanswered question is the nature of the functional consequences of IMPDH1 mutations that lead to retinal degeneration. Recent research shows that IMPDH binds single-stranded nucleic acids, suggesting another possible mechanism by which IMPDH1 mutations could cause retinal disease.9 In vitro and in vivo assays demonstrate that several IMPDH species, including human types I and II, bind random pools of single-stranded nucleotides via a protein subdomain composed of two CBS domains, named for homologous domains in cystathionine β-synthase (CBS). IMPDH can be found in both the cytoplasm and the nucleus of cultured cells; it binds both RNA and single-stranded DNA but does not bind to cognate RNA.9 Additional studies show that the Arg224Pro and Asp226Asn adRP mutations affect the affinity and the specificity of IMPDH1 nucleic acid binding and suggest that testing this functional property will assist in determining pathogenicity of novel IMPDH1 variants.8 In this study, we surveyed a large population of patients with adRP to determine the range and the frequency of IMPDH1 mutations. The clinical heterogeneity of mutations in genes associated with retinal degeneration has been demonstrated many times.10-13 Therefore, we also analyzed patients with a variety of other inherited retinal degenerations, specifically autosomal recessive RP (arRP), macular degeneration (MD), and Leber congenital amaurosis (LCA), to investigate the possibility that mutations in IMPDH1 cause alternate phenotypes. The enzymatic activity and nucleic acid binding properties of each novel IMPDH1 protein variant identified in these patients were also determined and used to infer pathogenicity.
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Mutations in the inosine monophosphate dehydrogenase 1 gene (IMPDH1) cause the RP10 form of autosomal dominant retinitis pigmentosa
Human molecular genetics, 2002Co-Authors: Sara J Bowne, Lori S Sullivan, Dianna K. Hughbanks-wheaton, David G. Birch, Susan H. Blanton, Constance L. Cepko, Seth Blackshaw, John R. Heckenlively, Stephen P DaigerAbstract:Autosomal dominant retinitis pigmentosa (adRP) is a heterogeneous set of progressive retinopathies caused by several distinct genes. One locus, the RP10 form of adRP, maps to human chromosome 7q31.1 and may account for 5-10% of adRP cases among Americans and Europeans. We identified two American families with the RP10 form of adRP by linkage mapping and used these families to reduce the linkage interval to 3.45 Mb between the flanking markers D7S686 and RP-STR8. Sequence and transcript analysis identified 54 independent genes within this region, at least 10 of which are retinal-expressed and thus candidates for the RP10 gene. A screen of retinal transcripts comparing retinas from normal mice to retinas from crx-/crx- knockout mice (with poorly differentiated photoreceptors) demonstrated a 6-fold reduction in one candidate, inosine monophosphate dehydrogenase 1 (IMPDH1; EC 1.1.1.205). Since many of the genes known to cause retinitis pigmentosa are under CRX control in photoreceptors, IMPDH1 became a high-priority candidate for mutation screening. DNA sequencing of affected individuals from the two American RP10 families revealed a GAC-->AAC transition in codon 226 substituting an asparagine for an aspartic acid in both families. The identical mutation was also found in a British RP10 family. The Asp226Asn missense mutation is present in all affected individuals tested and absent from unaffected controls. The aspartic acid at codon 226 is conserved in all IMPDH genes, in all species examined, including bacteria, suggesting that this mutation is highly deleterious. Subsequent screening of probands from 60 other adRP families revealed an additional family with this mutation, confirming its association with retinitis pigmentosa and the relatively high frequency of this mutation. Another IMPDH1 substitution, Val268Ile, was also observed in this cohort of patients but not in controls. IMPDH1 is a ubiquitously expressed enzyme, functioning as a homotetramer, which catalyzed the rate-limiting step in de novo synthesis of guanine nucleotides. As such, it plays an important role in cyclic nucleoside metabolism within photoreceptors. Several classes of drugs are known to affect IMPDH isoenzymes, including nucleotide and NAD analogs, suggesting that small-molecule therapy may be available, one day, for RP10 patients.
