Inosine monophosphate dehydrogenase (IMPDH) catalyses the key step in de novo guanine nucleotide biosynthesis at the branch point of GTP and ATP production. Mammals have two ubiquitous, catalytically indistinguishable isoforms, IMPDH type I and type II, and these are considered functionally interchangeable. Each contains a Bateman domain known to serve as energy-sensing / allosteric regulatory modules in a range of unrelated proteins. Mutations in the Bateman domain of type I, which do not affect catalytic activity, cause the retina-degenerative disease, retinitis pigmentosa (RP). The central hypothesis of this thesis is that IMPDH is regulated. In particular, that regulation occurs in an isoform specific manner and that mutations causal to RP affect enzyme regulation. Here we have visualised, including in real-time, the redistribution or clustering of IMPDH into linear macrostructures in a time-dependent manner which appeared to be intimately associated with changes in intracellular nucleotide levels. Data presented suggest the significance of IMPDH clustering is unlikely to be associated with substrate channelling, via interaction with other proteins in the de novo biosynthesis pathway, or enhanced protein stability. Although both isoforms responded similarly to fluctuations in intracellular nucleotide levels, type I had a higher propensity to spontaneously cluster into macrostructures compared to type II. This propensity to cluster was found to be conferred by the N-terminal 244 amino acids, which includes the Bateman domain, using a series of type I / type II chimera proteins. A comparative and novel approach revealed isoform-specific purine nucleotide binding characteristics. Type I bound ATP and type II bound AMP, via a mechanism involving the Bateman domain, resulting in conformational changes in IMPDH. This nucleotide binding was not associated with allosteric activation of IMPDH catalytic activity. The RP-causing mutation, R224P, abolished ATP binding and this correlated with an altered propensity to cluster. Collectively these data (i) show IMPDH distribution is regulated by the intracellular environment (ii) demonstrate that the IMPDH isoforms are modulated in a differential manner by AMP and ATP by a mechanism involving the Bateman domain, (iii) indicate communication between the Bateman domain and the active site and (iv) demonstrate that a RP-causing mutation compromises such regulation. From a broader perspective, this work raises the possibility that the nucleotide sensing properties of the Bateman domain in IMPDH serve to regulate IMPDH and co-ordinate nucleotide homeostasis, thereby giving rise to cellular plasticity in an isoform-specific manner to meet the requirements of the cellular environment.

Proefschrift - Amsterdam. / "Geraadpleegde literatuur": p. [90]-91.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24087号 / 医博第4863号 / 新制||医||1059(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 近藤 尚己, 教授 山本 洋介, 教授 辻川 明孝 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

hPrp8 is an essential pre-mRNA splicing factor. This highly conserved protein is a component of the U5 small ribonucleoprotein particle (U5 snRNP), which constitutes one of the building blocks of the spliceosome. hPrp8 acts as a key regulator of spliceosome activation and interacts directly with U5 snRNA and with the regions of pre-mRNA that are involved in the transesterification reactions during splicing. Mutations in hPrp8 have been shown to cause an autosomal dominant form of retinitis pigmentosa (RP), an inherited disease leading to progressive degeneration of retina. In this study, we analyzed the effects of the RP-associated mutations on the function of hPrp8. Using BAC recombineering, we created mutant variants of hPrp8-GFP construct and we generated stable cell lines expressing the recombinant proteins. The mutant proteins were expressed and localized to the nucleus. However, one of the missense mutations affected the localization and stability of hPrp8. Further experiments suggested that RP-associated mutations affect the ability of hPrp8 to interact with other components of the U5 snRNP and with pre-mRNA. We further studied the biogenesis of U5 snRNP. We depleted hPrp8 by siRNA to interfere with U5 snRNP assembly and we observed that the incompletely assembled U5 snRNPs accumulate in...

Retinose Pigmentar (RP) é uma doença hereditária que conduz progressivamente à cegueira. Mais de 150 mutações da rodopsina associadas à RP foram descritas, e causam a alteração da sua conformação. Esta tese testou a hipótese de que pequenas moléculas auxiliam na formação da rodopsina e/ou reduzem a morte dos fotorreceptores. As mutações da RP, N15S e P23H, revelaram diferenças quanto às características e gravidade devido à má-formação das proteínas mutantes. Ligação de pequenas moléculas (retinóides, íons metálicos, clorofilas e antocianinas) à rodopsina foi demonstrada in vitro. O derivado da clorofila, Ce6, mostrou-se mais efetivo, conferindo maior estabilidade e foi então testado em ratos submetidos à degeneração por luz ou em modelos de RP (P23H e S334ter). Observou-se uma proteção contra a degeneração por luz e uma significante diminuição da degeneração no P23H. Em contraste, Ce6 causou um aumento na degeneração dos fotorreceptores do S334ter. Finalmente, resultados clínicos, bioquímicos e in vivo foram comparados e mostraram estar altamente relacionados. / Retinitis Pigmentosa (RP) is an inherited disease that progressively leads to blindness. More than 150 mutations associated with RP are known in rhodopsin, causing its misfolding. This thesis tested the hypothesis that small molecules can rescue folded rhodopsin and/or reduce photoreceptor cell death. RP mutations, N15S and P23H, revealed differences in characteristics and severity of misfolding of the mutant proteins. Binding of small molecule classes (retinals, metal ions, chlorophylls and anthocyanins) to rhodopsin was demonstrated in vitro. The chlorophyll derivative, Ce6, was most effective in conferring stability and therefore tested in rats subjected to light-damage and RP rat models, P23H and S334ter. Protection against the light-induced retinal degeneration and more importantly a significant slowing of the photoreceptor degeneration rate in the P23H rat were observed. In contrast, Ce6 increased photoreceptor degeneration in the S334ter rat. Finally, clinical, biochemical and in vivo rat data were compared and it was found to be highly correlated.

The term 'Retinitis pigmentosa' (RP) represents a group of inherited, late-onset diseases characterised by progressive retinal degeneration due to photoreceptor death. Mutations in the RP2 gene are found in 7-18% of patients with X-linked RP, one of the most severe forms. The RP2 gene product is a membrane-associated protein which encompasses two distinct domains. The N-terminal domain is well characterised as possessing GTPase-activating protein (GAP) activity towards the small GTPase ARL3 and thus regulate the transport of lipid-modified proteins within the photoreceptor cell. However, it is not known if the loss of this particular function of RP2 is the sole reason that causes the disease, while the role of the protein's C-terminus remains unknown. This thesis focuses on the characterisation of two novel protein-protein interactions of RP2 with the aim to investigate novel roles of the protein. Firstly, evidence is provided that a highly-conserved cluster of RP2 residues that span both the N- and C-terminus participate in direct interaction with Osteoclast-stimulating factor 1 (OSTF1). Two hypotheses are explored about the potential role of the complex in SRC-mediated RP2 phosphorylation and the regulation of cell motility. Secondly, the catalytic subunit of DNA-dependent protein kinase (DNA PK) is identified as a novel interaction partner of RP2 in cultured cells. The two proteins are shown to co-localise in the nuclear and membrane compartments of a retinal-derived cell line and might engage in a kinase-substrate relationship. So far, no evidence was found that RP2 participates in the canonical function of DNA PK which is the regulation of DNA double-stranded breaks. Finally, the CRISPR/Cas9 genome editing method was applied on zebrafish embryos to generate a novel vertebrate animal model for the loss of RP2 function. One out of three different zebrafish lines with rp2 mutations was shown by histology to have mild late-onset thinning of the photoreceptor outer segments. The present thesis reports previously unexplored aspects of RP2's function and will, therefore, contribute to understanding the molecular mechanisms that underlie RP. Moreover, this thesis will contribute to the discussion about the usefulness of zebrafish as an RP model.

Retinitis Pigmentosa (RP) är en ögonsjukdom som leder till blindhet, sjukdomsförloppet varierar från individ till individ och det finns för närvarande ingen botande behandling. Att utveckla en synskada successivt och bli tvungen att anpassa sig efter en ny vardag skapar osäkerhet och ett stort lidande. Hjälpmedel som underlättar vardagen mottages motvilligt och den synskadade förnekar vanligtvis sin situation. Som sjuksköterska är det viktigt att ha förståelse för den synskadades situation och lidande för att kunna skapa en god vårdrelation. Problemet är att det finns lite forskning inom området, vilket leder till upprepade missförstånd och ett vårdlidande för patienten. Syftet med studien är att beskriva hur det är att leva med vetskapen om att ha en sjukdom som leder till blindhet och hur den förändrande vardagen upplevs och hanteras. Vi har valt att analysera biografier för att få patientens beskrivning av sin subjektiva upplevelse och den metod som Dahlborg Lyckhage beskriver har stått till grund för analysen. I analysen framkom fyra huvudteman med underteman som ligger till grund för resultatet. De fyra huvudtemana är: svårigheter med att acceptera sin sjukdom, de första stegen till hantering, att bli sedd som blind och att bli vän med sin sjukdom. Vi har funnit liknande resultat i andras studier och resultatet upplevs som relevant för den vård som sjuksköterskan praktiserar. Det är viktigt att synliggöra och uppmärksamma dessa personers livsvärld då RP är en vanlig ögonsjukdom och det är omöjligt att veta när en synskadad patient kommer in genom dörren till avdelningen. / <p>Program: Sjuksköterskeutbildning</p><p>Uppsatsnivå: C</p>

Inherited retinal degenerations are a group of disorders characterised by great genetic heterogeneity. Clinically, they can be divided into two large groups of diseases, those associated with night blindness, e.g. retinitis pigmentosa (RP), and those with macular malfunction, e.g. cone/cone-rod dystrophy (COD/CORD). This thesis is focused on finding the genetic basis of disease in families with autosomal dominant COD, autosomal dominant RP, and Bothnia dystrophy (BD), a regional variant of RP.   A variant of COD was previously mapped to 17p12-p13 in a family from northern Sweden. One additional family originating from the same geographical area was included in fine mapping of this chromosome region. Using 12 microsatellite markers in linkage and haplotype analysis, the region was refined from 26.9 to 14.3 cM. A missense mutation, Q626H, in an evolutionarily conserved region of PITPNM3, phosphatidylinositol transfer membrane-associated protein, was identified. The mutation segregated with the disease in both families and was absent from normal control chromosomes. PITPNM3 is a human homologue of the Drosophila retinal degeneration (rdgB) protein, which is highly expressed in the retina and has been proposed to be required for membrane turnover of photoreceptor cells. With the intention of establishing the global impact that PITPNM3 has on retinal degenerations 165 DNA samples from COD and CORD patients were obtained from Denmark, Germany, the UK, and USA and screened for mutations. The Q626H mutation found in the Swedish families was also found in one British family and a novel Q342P variant was detected in a German patient. In addition, two intronic variants were identified: c.900+60C&gt;T and c.901-45G&gt;A. Thus, we concluded that mutations in PITPNM3 represent a rare cause of COD worldwide. In two large families from northern Sweden showing autosomal dominant RP with reduced penetrance, the disease locus was mapped using genome-wide linkage analysis to 19q13.42 (RP11). Since mutation screening of eight genes on 19q13.42 revealed no mutations, multiplex ligation-dependent probe amplification (MLPA) was used to screen for large genomic abnormalities in PRPF31, RHO, RP1, RPE65, and IMPDH1. A large deletion spanning 11 exons of PRPF31 and three genes upstream was identified. Using long-range PCR, the breakpoints of the deletion were identified and the size of the deletion was determined to encompass almost 59 kb. BD is an autosomal recessive type of RP with high prevalence in northern Sweden. The disease is associated with a c.700C&gt;T mutation in RLBP1. In a screening of recessive RP in northern Sweden, 67 patients were found to be homozygous for c.700C&gt;T and 10 patients were heterozygous. An evaluation with arrayed primer extension (APEX) technology revealed a second mutation, c.677T&gt;A, in RLBP1 giving rise to compound heterozygosity in these patients. In addition, a c.40C&gt;T exchange in CAIV was detected in a patient with BD and in 143 healthy blood donors. The c.40C&gt;T substitution in CAIV has been reported to cause autosomal dominant RP in South African families with European ancestry. However, in the population of northern Sweden it appears to be a benign polymorphism. In summary, a first mutation in PITPNM3, encoding a human homologue of the Drosophila retinal degeneration protein, was detected in two large families with COD. A large deletion in PRPF31 was discovered in two families with autosomal dominant RP showing reduced penetrance and in 10 patients BD was shown to be caused by two allelic mutations in RLBP1.

Thesis (Ph.D.)--University of Alberta, 2010. / A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy, Department of Biochemistry. Title from pdf file main screen (viewed on February 12, 2010). Includes bibliographical references.

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