Polyglutamine and Polyalanine Tracts Are Enriched in Transcription Factors of Plants

Polyglutamine (polyQ) tracts have been studied extensively for their roles in a number of human diseases such as Huntington\u27s or different Ataxias. However, it has also been recognized that polyQ tracts are abundant and may have important functional and evolutionary roles. Especially the association of polyQ and also polyalanine (polyA) tracts with transcription factors and their activation activity has been noted. While a number of examples for this association have been found for proteins from opisthokonts (animals and fungi), only a few studies exist for polyQ and polyA stretches in plants, and systematic investigations of the significance of these repeats in plant transcription factors are scarce. Here, we analyze the abundance and length of polyQ and polyA stretches in the conceptual proteomes of six plant species and examine the connection between polyQ and polyA tracts and transcription factors of the repeat-containing proteins. We show that there is an association of polyQ stretches with transcription factors in plants. In grasses, transcription factors are also significantly enriched in polyA stretches. While there is variation in the abundance, length, and association with certain functions of polyQ and polyA stretches between different species, no general differences in the evolution of these repeats could be observed between plants and opisthokonts

A Variable Polyglutamine Repeat Affects Subcellular Localization and Regulatory Activity of a Populus ANGUSTIFOLIA Protein.

Polyglutamine (polyQ) stretches have been reported to occur in proteins across many organisms including animals, fungi and plants. Expansion of these repeats has attracted much attention due their associations with numerous human diseases including Huntington's and other neurological maladies. This suggests that the relative length of polyQ stretches is an important modulator of their function. Here, we report the identification of a Populus C-terminus binding protein (CtBP) ANGUSTIFOLIA (PtAN1) which contains a polyQ stretch whose functional relevance had not been established. Analysis of 917 resequenced Populus trichocarpa genotypes revealed three allelic variants at this locus encoding 11-, 13- and 15-glutamine residues. Transient expression assays using Populus leaf mesophyll protoplasts revealed that the 11Q variant exhibited strong nuclear localization whereas the 15Q variant was only found in the cytosol, with the 13Q variant exhibiting localization in both subcellular compartments. We assessed functional implications by evaluating expression changes of putative PtAN1 targets in response to overexpression of the three allelic variants and observed allele-specific differences in expression levels of putative targets. Our results provide evidence that variation in polyQ length modulates PtAN1 function by altering subcellular localization

Biology and characterisation of polyalanine as an emerging pathological marker

Dix-huit maladies humaines graves ont jusqu'ici été associées avec des expansions de trinucléotides répétés (TNR) codant soit pour des polyalanines (codées par des codons GCN répétés) soit pour des polyglutamines (codées par des codons CAG répétés) dans des protéines spécifiques. Parmi eux, la dystrophie musculaire oculopharyngée (DMOP), l’Ataxie spinocérébelleuse de type 3 (SCA3) et la maladie de Huntington (MH) sont des troubles à transmission autosomale dominante et à apparition tardive, caractérisés par la présence d'inclusions intranucléaires (IIN). Nous avons déjà identifié la mutation responsable de la DMOP comme étant une petite expansion (2 à 7 répétitions supplémentaires) du codon GCG répété du gène PABPN1. En outre, nous-mêmes ainsi que d’autres chercheurs avons identifié la présence d’événements de décalage du cadre de lecture ribosomique de -1 au niveau des codons répétés CAG des gènes ATXN3 (SCA3) et HTT (MH), entraînant ainsi la traduction de codons répétés hybrides CAG/GCA et la production d'un peptide contenant des polyalanines. Or, les données observées dans la DMOP suggèrent que la toxicité induite par les polyalanines est très sensible à leur quantité et leur longueur. Pour valider notre hypothèse de décalage du cadre de lecture dans le gène ATXN3 dans des modèles animaux, nous avons essayé de reproduire nos constatations chez la drosophile et dans des neurones de mammifères. Nos résultats montrent que l'expression transgénique de codons répétés CAG élargis dans l’ADNc de ATXN3 conduit aux événements de décalage du cadre de lecture -1, et que ces événements sont néfastes. À l'inverse, l'expression transgénique de codons répétés CAA (codant pour les polyglutamines) élargis dans l’ADNc de ATXN3 ne conduit pas aux événements de décalage du cadre de lecture -1, et n’est pas toxique. Par ailleurs, l’ARNm des codons répétés CAG élargis dans ATXN3 ne contribue pas à la toxicité observée dans nos modèles. Ces observations indiquent que l’expansion de polyglutamines dans nos modèles drosophile et de neurones de mammifères pour SCA3 ne suffit pas au développement d'un phénotype. Par conséquent, nous proposons que le décalage du cadre de lecture ribosomique -1 contribue à la toxicité associée aux répétitions CAG dans le gène ATXN3. Pour étudier le décalage du cadre de lecture -1 dans les maladies à expansion de trinucléotides CAG en général, nous avons voulu créer un anticorps capable de détecter le produit présentant ce décalage. Nous rapportons ici la caractérisation d’un anticorps polyclonal qui reconnaît sélectivement les expansions pathologiques de polyalanines dans la protéine PABPN1 impliquée dans la DMOP. En outre, notre anticorps détecte également la présence de protéines contenant des alanines dans les inclusions intranucléaires (IIN) des échantillons de patients SCA3 et MD.Eighteen severe human diseases have thus far been associated with trinucleotide repeat (TNR) expansions coding for either polyalanine (encoded by a GCN repeat tract) or polyglutamine (encoded by a CAG repeat tract) in specific proteins. Among them, oculopharyngeal muscular dystrophy (OPMD), spinocerebellar ataxia type-3 (SCA3), and Huntington’s disease (HD) are late-onset autosomal-dominant disorders characterised by the presence of intranuclear inclusions (INIs). We have previously identified the OPMD causative mutation as a small expansion (2 to 7) of a GCG repeat tract in the PABPN1 gene. In addition, we and others have reported the occurrence of -1 ribosomal frameshifting events in expanded CAG repeat tracts in the ATXN3 (SCA3) and HTT (HD) genes, which result in the translation of a hybrid CAG/GCA repeat tract and the production of a polyalanine-containing peptide. Data from OPMD suggests that polyalanine-induced toxicity is very sensitive to the dosage and length of the alanine stretch. To validate our ATXN3 -1 frameshifting hypothesis in animal models, we set out to reproduce our findings in Drosophila and mammalian neurons. Our results show that the transgenic expression of expanded CAG repeat tract ATXN3 cDNA led to -1 frameshifting events, and that these events are deleterious. Conversely, the expression of polyglutamine-encoding expanded CAA repeat tract ATXN3 cDNA was neither frameshifted nor toxic. Furthermore, expanded CAG repeat tract ATXN3 mRNA does not contribute to the toxicity observed in our models. These observations indicate that expanded polyglutamine repeats in Drosophila and mammalian neuron models of SCA3 are insufficient for the development of a phenotype. Hence, we propose that -1 ribosomal frameshifting contributes to the toxicity associated with CAG repeat tract expansions in the ATXN3 gene. To further investigate ribosomal frameshifting in expanded CAG repeat tract diseases, we sought to create an antibody capable of detecting the frameshifted product. Here we report the characterization of a polyclonal antibody that selectively recognizes pathological expansions of polyalanine in the protein implicated in OPMD, PABPN1. Furthermore, our antibody also detects the presence of alanine proteins in the intranuclear inclusions (INIs) of SCA3 and HD patient samples

Tandem amino acid repeats in the green anole (Anolis carolinensis) and other squamates may have a role in increasing genetic variability

Statistics of the other amino acid repeat types in the green anole proteome (supplementary table for Table 2 ). (DOCX 67 kb

Chaperones and proteases: Cellular fold-controlling factors of proteins in neurodegenerative diseases and aging

The formation of toxic protein aggregates is a common denominator to many neurode generative diseases and aging. Accumulation of toxic, possibly infectious protein aggregates induces a cascade of events, such as excessive inflammation, the production of reactive oxygen species, apoptosis and neuronal loss. A network of highly conserved molecular chaperones and of chaperone-related proteases controls the fold-quality of proteins in the cell. Most molecular chaperones can passively prevent protein aggregation by binding misfolding inter mediates. Some molecular chaperones and chaperone-related proteases, such as the proteasome, can also hydrolyse ATP to forcefully convert stable barmful protein aggregates into harmless natively refoldable, or protease-degradable, polypeptides. Molecular chaperones and chaperone-related proteases thus control the delicate balance between natively folded functional proteins and aggregation-prone misfolded proteins, which may form during the lifetime and lead to cell death. Abundant data now point at the molecular chaperones and the proteases as major clearance mechanisms to remove toxic protein aggregates from cells, delaying the onset and the outcome of protein-misfolding diseases. Therapeutic approaches include treatments and drugs that can specifically induced and sustain a strong chaperone and protease activity in cells and tissues prone to toxic protein aggregation

RNAi based allele-specific silencing of the disease-causing gene in black South African patients with SCA7

Includes abstract.Includes bibliographical references (leaves 118-133).The polyglutamine disorders are a subgroup of inherited neurodegenerative disorders with a common mutation which confers toxicity via a polyglutamine tract in the protein leading ultimately to various forms of neurodegeneration. One of these disorders, spinocerebellar ataxia 7 (SCA7) exists at a higher frequency in South Africa, than elsewhere in the world, and a founder effect has been demonstrated in South Africa, such that every patient tested thus far is linked to a common ancestor. The manipulation of RNA interference (RNAi) has been used with increasing success to selectively knockdown the expression of disease-causing genes at the RNA level. Thus, the possibility of applying this method to SCA7 in South Africa was considered. However, the wild-type allele of ataxin-7 is likely to be necessary for cellular function therefore a form of allele-specific silencing is required, such as a SNP linked to the mutation

The role of epigenetic factors in the pathogenesis of familial X-linked mental retardation (XLMR)

Mental retardation (MR) is a handicap with severe implications not only for thosethat suffer from this disability, but also for their families, society and the welfaresystems which support them. A large proportion of these individuals are afflictedwith the X-linked form of the condition. To date a total of 87 genes have beenimplicated in the pathogenesis of X-linked mental retardation (XLMR)

Insights into pathology and neurodegeneration features in a transgenic mouse model of Machado-Joseph disease

Dissertação de mestrado em Genética MolecularMachado-Joseph disease (MJD), also known as Spinocerebellar Ataxia 3 (SCA3), is the most common autosomal dominant ataxia worldwide, and is caused by a CAG repeat expansion within the coding region of the ATXN3 gene. The clinical variability of the disease phenotype as well as the age of onset depend on the length of the expanded repeat. The anticipation phenomenon is most frequently associated with repeat expansions in paternal transmission. MJD patients with a repeat expansion above 44 CAGs in the ATXN3 gene present cytoplasmic and/or intranuclear ataxin-3 aggregates and neuronal cell loss in specific areas of the brain. However, some questions remains unanswered in this disease: why only some subpopulations of neurons are affected, although ataxin- 3 is everywhere; what underlies this selective neuronal vulnerability; are these neurons dysfunctional or dying? In an attempt to address these issues, we took advantage of studying a cDNA transgenic mouse model (CMVMJD) expressing the mutant human ataxin-3 under the regulation of the CMV promoter (pCMV), previously generated in our lab. This transgenic mouse model shows an important overlap with genetic and clinical features of MJD, namely genetic instability of the expanded CAG repeat and a motor impairment phenotype. In this work, we performed an extensive pathological analysis of MJD mouse brains, that revealed a significant atrophy in the thalamus and in the dentate neurons. Increased GFAP immunostaining with reactive astrocytes was observed in the vestibular nuclei and substantia nigra of transgenic mice. Regarding cell death, we have searched for evidence of different cell death types (apoptosis and necrosis) by TUNEL assay, caspase-3 analysis and Fluoro-Jade B staining. We did not find any differences between wild-type and MJD transgenic mice, suggesting that probably the affected neurons are not dying, at least by apoptosis or necrosis, instead, they might just be dysfunctional. We also analysed of the somatic mosaicism in neuronal and non-neuronal tissues through aging revealed a significant increase in the mosaicism index of specific brain regions such as the pons, substantia nigra, cerebellar cortex, hipocampus, striatum, deep cerebellar nuclei and hypothalamus with age. However, there was no correlation between the extent of the mosaicism and the pathological involvement of a given region. The results allow us to conclude that the pCMVMJD94 mouse is a good model to study the pathogenic mechanisms of MJD, mimicking an early stage of the disease.A doença de Machado-Joseph, também conhecida por Ataxia Espinocerebelosa tipo 3 (SCA3), é a ataxia autossómica dominante mais comum em todo o mundo, causada por uma repetição de CAGs na região codificante do gene ATXN3. A variabilidade no fenótipo da doença assim como a idade de surgimento dos sintomas depende no tamanho da repetição expandida. O fenómeno de antecipação está mais frequentemente associado a transmissões paternas. Pacientes com DMJ com expansões acima de 44 CAGs revelam agregados citoplasmáticos e /ou nucleares e perda neuronal em áreas do cérebro específicas. Algumas questões chave permanecem por esclarecer. Por exemplo, nesta doença apenas algumas subpopulações de neurónios são afectadas, embora a ataxina-3 seja expressa em todas as áreas. O que está por detrás desta vulnerabilidade neuronal selectiva? Estarão estes neurónios a morrer ou estarão disfuncionais? Numa tentativa de responder a estas perguntas, utilizámos um modelo em ratinho que expressa o cDNA da ataxina-3 humana mutada, sob a regulação do promotor CMV (pCMV), previamente gerado no nosso laboratório. Neste trabalho, fizemos uma extensa análise patológica de cérebros de ratinho DMJ que revelaram uma atrofia relevante no tálamo e núcleos denteados. Também se observou um aumento da proteína GFAP, revelando um aumento da reactividade dos astrócitos, nos núcleos vestibular e substantia nigra dos ratinhos transgénicos. Também fizemos um rastreio de diferentes tipos de morte celular (apoptose e necrose) por TUNEL, análise da activação da caspase-3 (imunohistoquímica e western-blot) e coloração com Fluoro-Jade B. Não encontrámos diferenças significativas entre ratinhos do tipo selvagem e transgénicos, sugerindo que provavelmente estes neurónios não estarão a morrer, pelo menos por apoptose ou necrose, estando possivelmente disfuncionais. Para além disso, a repetição de CAG expandida, variou em mais de 50% das transmissões nos ratinhos transgénicos, com expansões típica transmitidas em meioses paternas e contracções em maternas. A análise do mosaicismo somático em tecidos neuronais e periféricos, durante o envelhecimento, revelou um aumento no índex de mosaicismo em regiões específicas do cérebro e mostrou ser dependente da idade. Este ratinho transgénico apresenta características genéticas e clínicas importantes que se sobrepõem às da DMJ, nomeadamente instabilidade intergeracional da expansão de CAGs, características patológicas da doença (astrogliose e neurónios atrofiados, nas regiões relevantes) e um fenótipo de descoordenação motora. Estes resultados permitiram-nos concluir que o ratinho pCMVMJD94 é um bom modelo para estudar os mecanismos patogénicos da DMJ, mimetizando estadios precoces da doença