An automatic method for assessing structural importance of amino acid positions

Background: A great deal is known about the qualitative aspects of the sequence-structure relationship, for example that buried residues are usually more conserved between structurally similar homologues, but no attempts have been made to quantitate the relationship between evolutionary conservation at a sequence position and change to global tertiary structure. In this paper we demonstrate that the Spearman correlation between sequence and structural change is suitable for this purpose. Results: Buried residues, bends, cysteines, prolines and leucines were significantly more likely to occupy positions highly correlated with structural change than expected by chance. Some buried residues were found to be less informative than expected, particularly residues involved in active sites and the binding of small molecules. Conclusion: The correlation-based method generates predictions of structural importance for superfamily positions which agree well with previous results of manual analyses, and may be of use in automated residue annotation piplines. A PERL script which implements the method is provided

Potential protein activity modifications of amino acid variants in the human transcriptome

Background: The occurrence of widespread RNA and DNA sequence differences in the human transcriptome was reported in 2011. Similar findings were described in a second independent publication on personal omics profiling investigating the occurrence of dynamic molecular and related medical phenotypes. The suggestion that the RNA sequence variation was likely to affect disease susceptibility prompted us to investigate with a range of algorithms the amino acid variants reported to be present in the identified peptides to determine if they might be disease-causing. Results: The predictive qualities of the different algorithms were first evaluated by using nonsynonymous single-base nucleotide polymorphism (nsSNP) datasets, using independently established data on amino acid variants in several proteins as well as data obtained by mutational mapping and modelling of binding sites in the human serotonin transporter protein (hSERT). Validation of the used predictive algorithms was at a 75% level. Using the same algorithms, we found that widespread RNA and DNA sequence differences were predicted to impair the function of the peptides in over 57% of cases. Conclusions: Our findings suggest that a proportion of edited RNAs which serve as templates for protein synthesis is likely to modify protein function, possibly as an adaptive survival mechanism in response to environmental modifications

A graph-theoretic approach for classification and structure prediction of transmembrane β-barrel proteins

BACKGROUND: Non-synonymous coding SNPs (nsSNPs) that are associated to disease can also be related with alterations in protein stability. Computational methods are available to predict the effect of single amino acid substitutions (SASs) on protein stability based on a single folded structure. However, the native state of a protein is not unique and it is better represented by the ensemble of its conformers in dynamic equilibrium. The maintenance of the ensemble is essential for protein function. In this work we investigated how protein conformational diversity can affect the discrimination of neutral and disease related SASs based on protein stability estimations. For this purpose, we used 119 proteins with 803 associated SASs, 60% of which are disease related. Each protein was associated with its corresponding set of available conformers as found in the Protein Conformational Database (PCDB). Our dataset contains proteins with different extensions of conformational diversity summing up a total number of 1023 conformers. RESULTS: The existence of different conformers for a given protein introduces great variability in the estimation of the protein stability (\u394\u394G) after a single amino acid substitution (SAS) as computed with FoldX. Indeed, in 35% of our protein set at least one SAS can be described as stabilizing, destabilizing or neutral when a cutoff value of \ub12 kcal/mol is adopted for discriminating neutral from perturbing SASs. However, when the \u394\u394G variability among conformers is taken into account, the correlation among the perturbation of protein stability and the corresponding disease or neutral phenotype increases as compared with the same analysis on single protein structures. At the conformer level, we also found that the different conformers correlate in a different way to the corresponding phenotype. CONCLUSIONS: Our results suggest that the consideration of conformational diversity can improve the discrimination of neutral and disease related protein SASs based on the evaluation of the corresponding Gibbs free energy change

SDM—a server for predicting effects of mutations on protein stability and malfunction

The sheer volume of non-synonymous single nucleotide polymorphisms that have been generated in recent years from projects such as the Human Genome Project, the HapMap Project and Genome-Wide Association Studies means that it is not possible to characterize all mutations experimentally on the gene products, i.e. elucidate the effects of mutations on protein structure and function. However, automatic methods that can predict the effects of mutations will allow a reduced set of mutations to be studied. Site Directed Mutator (SDM) is a statistical potential energy function that uses environment-specific amino-acid substitution frequencies within homologous protein families to calculate a stability score, which is analogous to the free energy difference between the wild-type and mutant protein. Here, we present a web server for SDM (http://www-cryst.bioc.cam.ac.uk/~sdm/sdm.php), which has obtained more than 10 000 submissions since being online in April 2008. To run SDM, users must upload a wild-type structure and the position and amino acid type of the mutation. The results returned include information about the local structural environment of the wild-type and mutant residues, a stability score prediction and prediction of disease association. Additionally, the wild-type and mutant structures are displayed in a Jmol applet with the relevant residues highlighted

Using structural bioinformatics to investigate the impact of non synonymous SNPs and disease mutations: scope and limitations

BACKGROUND: Linking structural effects of mutations to functional outcomes is a major issue in structural bioinformatics, and many tools and studies have shown that specific structural properties such as stability and residue burial can be used to distinguish neutral variations and disease associated mutations. RESULTS: We have investigated 39 structural properties on a set of SNPs and disease mutations from the Uniprot Knowledge Base that could be mapped on high quality crystal structures and show that none of these properties can be used as a sole classification criterion to separate the two data sets. Furthermore, we have reviewed the annotation process from mutation to result and identified the liabilities in each step. CONCLUSION: Although excellent annotation results of various research groups underline the great potential of using structural bioinformatics to investigate the mechanisms underlying disease, the interpretation of such annotations cannot always be extrapolated to proteome wide variation studies. Difficulties for large-scale studies can be found both on the technical level, i.e. the scarcity of data and the incompleteness of the structural tool suites, and on the conceptual level, i.e. the correct interpretation of the results in a cellular context.status: publishe

Prediction of the responsiveness to pharmacological chaperones: lysosomal human alpha-galactosidase, a case of study

<p>Abstract</p> <p>Background</p> <p>The pharmacological chaperones therapy is a promising approach to cure genetic diseases. It relies on substrate competitors used at sub-inhibitory concentration which can be administered orally, reach difficult tissues and have low cost. Clinical trials are currently carried out for Fabry disease, a lysosomal storage disorder caused by inherited genetic mutations of alpha-galactosidase. Regrettably, not all genotypes respond to these drugs.</p> <p>Results</p> <p>We collected the experimental data available in literature on the enzymatic activity of ninety-six missense mutants of lysosomal alpha-galactosidase measured in the presence of pharmacological chaperones. We associated with each mutation seven features derived from the analysis of 3D-structure of the enzyme, two features associated with their thermo-dynamic stability and four features derived from sequence alone. Structural and thermodynamic analysis explains why some mutants of human lysosomal alpha-galactosidase cannot be rescued by pharmacological chaperones: approximately forty per cent of the non responsive cases examined can be correctly associated with a negative prognostic feature. They include mutations occurring in the active site pocket, mutations preventing disulphide bridge formation and severely destabilising mutations. Despite this finding, prediction of mutations responsive to pharmacological chaperones cannot be achieved with high accuracy relying on combinations of structure- and thermodynamic-derived features even with the aid of classical and state of the art statistical learning methods.</p> <p>We developed a procedure to predict responsive mutations with an accuracy as high as 87%: the method scores the mutations by using a suitable position-specific substitution matrix. Our approach is of general applicability since it does not require the knowledge of 3D-structure but relies only on the sequence.</p> <p>Conclusions</p> <p>Responsiveness to pharmacological chaperones depends on the structural/functional features of the disease-associated protein, whose complex interplay is best reflected on sequence conservation by evolutionary pressure. We propose a predictive method which can be applied to screen novel mutations of alpha galactosidase. The same approach can be extended on a genomic scale to find candidates for therapy with pharmacological chaperones among proteins with unknown tertiary structures.</p

A structural bioinformatics approach to the analysis of nonsynonymous single nucleotide polymorphisms (nsSNPs) and their relation to disease

The prediction of the effects of nonsynonymous single nucleotide polymorphisms (nsSNPs) on function depends critically on exploiting all information available on the three-dimensional structures of proteins. We describe software and databases for the analysis of nsSNPs that allow a user to move from SNP to sequence to structure to function. In both structure prediction and the analysis of the effects of nsSNPs, we exploit information about protein evolution, in particular, that derived from investigations on the relation of sequence to structure gained from the study of amino acid substitutions in divergent evolution. The techniques developed in our laboratory have allowed fast and automated sequence-structure homology recognition to identify templates and to perform comparative modeling; as well as simple, robust, and generally applicable algorithms to assess the likely impact of amino acid substitutions on structure and interactions. We describe our strategy for approaching the relationship between SNPs and disease, and the results of benchmarking our approach - human proteins of known structure and recognized mutation. &#169; 2007 Imperial College Pressope

Structure-aided detection of functional innovation in protein phylogenies

Detection of positive selection in proteins is both a common and powerful approach for investigating the molecular basis of adaptation. In this thesis, I explore the use of protein three- dimensional (3D) structure to assist in prediction of historical adaptations in proteins. Building on a method first introduced by Wagner (Genetics, 2007, 176: 2451–2463), I present a novel framework called Adaptation3D for detecting positive selection by integrating sequence, structural, and phylogenetic information for protein families. Adaptation3D identifies possible instances of positive selection by reconstructing historical substitutions along a phylogenetic tree and detecting branch-specific cases of spatially clustered substitution. The Adaptation3D method was capable of identifying previously characterized cases of positive selection in proteins, as demonstrated through an analysis of the pathogenesis-related protein 5 (PR-5) phylogeny. It was then applied on a phylogenomic scale in an analysis of thousands of vertebrate protein phylogenetic trees from the Selectome database. Adaptation3D’s reconstruction of historical mutations in vertebrate protein families revealed several evolutionary phenomena. First, clustered mutation is widespread and occurs significantly more often than that expected by chance. Second, numerous top-scoring cases of predicted positive selection are consistent with existing literature on vertebrate protein adaptation. Third, in the vertebrate lineage, clustered mutation has occurred disproportionately in proteins from certain families and functional categories such as zinc-finger transcription factors (TFs). Finally, by separating paralogous and orthologous lineages, it was found that TF paralogs display significantly elevated levels of clustered mutation in their DNA-binding sites compared to orthologs, consistent with historical DNA-binding specificity divergence in newly duplicated TFs. Ultimately, Adaptation3D is a powerful framework for reconstructing structural patterns of historical mutation, and provides important insights into the nature of protein adaptation

A Tale of Two SNPS: Polymorphism Analysis of Toll-like Receptor (TLR) Adapter Proteins: A Dissertation

The innate immune system is the first line of defense against invading pathogens. Recognition of microbial ligands by the innate immune system relies on germ-line encoded, evolutionarily conserved receptors called pattern recognition receptors (PRRs). Toll-like receptors (TLRs) are one such family of PRRs and are involved in innate defenses to a variety of microbes. At the core of TLR signaling pathways are Toll interleukin-1 receptor (TIR) domain containing adapter proteins. Much of the specificity of TLR pathways arise from the differential use of these adapter proteins. The TLR signaling cascade that ensues upon ligand recognition is marked by finely orchestrated molecular interactions between the receptor and the TIR domain containing adapter proteins, as well as various downstream kinases and effector molecules. Conserving the structural integrity of the TLR components is thus essential for maintaining a robust host defense system. Sometimes, changes in a protein can be brought about by single nucleotide polymorphisms (SNPs). Studies carried out in this thesis focus on polymorphisms in MyD88 adapter-like (Mal) and myeloid differentiation protein 88 (MyD88), two TIR domain-containing adapter proteins, which incidentally are also highly polymorphic. Mal is a 235 amino acid protein that is involved in TLR2 and TLR4 signaling. The known polymorphisms in the coding region of Mal were screened with an aim to identify SNPs with altered signaling potential. A TIR domain polymorphism, D96N, was found to be completely defective in TLR2 and TLR4 signaling. Immortalized macrophage-like cell lines expressing D96N have impaired cytokine production as well as NF-κB activation. The reason for this loss-of-function phenotype is the inability of Mal D96N to bind the downstream adapter MyD88, an event necessary for signaling to occur. Genotyping studies reveal a very low frequency of this polymorphism in the population. Similar SNP analysis was carried out in myeloid differentiation protein 88 (MyD88). MyD88 is a key signaling adapter in TLR signaling; critical for all TLR pathways except TLR3. In reporter assays, a death domain variant, S34Y, was found to be inactive. Importantly, in reconstituted macrophage-like cell lines derived from knockout mice, MyD88 S34Y was severely compromised in its ability to respond to all MyD88-dependent TLR ligands. S34Y mutant has a dramatically different localization pattern as compared to wild type MyD88. Unlike wild type MyD88, S34Y is unable to form distinct foci in the cells but is present diffused in the cytoplasm. IRAK4, a downstream kinase, colocalizes with MyD88 in these aggregates or “Myddosomes”. S34Y MyD88, however, is unable to assemble into Myddosomes, thus demonstrating that proper cellular localization of MyD88 is a feature required for MyD88 function. This thesis thus describes two loss‐of‐function polymorphisms in TLR adapter proteins Mal and MyD88. It sheds light not only on the structural aspects of signaling by these two proteins, but also has implications for the development of novel pharmaceutical agents

Bases moleculares da galactosémia clássica : pesquisa de novas estratégias terapêuticas

Tese de doutoramento, Farmácia (Biologia Celular e Molecular), Universidade de Lisboa, Faculdade de Farmácia, 2014Classic galactosemia is a potentially lethal disease of the galactose metabolism caused by a severe deficiency of GALT, the second enzyme of the Leloir pathway. This disorder is associated with mutations in the GALT gene, and displays an autosomal recessive pattern of inheritance. Although apparently asymptomatic at birth, affected infants start developing escalating symptoms after 1 to 2 weeks of galactose ingestion due to milk feeding. Upon implementation of a galactose-restricted diet, infants show rapid clinical improvement and seem to be almost miraculously cured; however, the long-term outcome is often disappointing, as most patients go on to develop severe complications notwithstanding strict compliance with the dietary therapy. Despite decades of intensive research, classic galactosemia pathophysiology is still largely unknown, resulting in a limited and poorly effective therapy. A comprehensive understanding of the molecular mechanisms underlying classic galactosemia potentially opens new therapeutic avenues, and prompted us to develop the present work. The first part of this thesis (chapter 1) comprises a general introduction with a review of the literature concerning the molecular and biochemical basis of classic galactosemia, followed by an overview of the current therapeutic approaches in genetic disorders – with a special focus on inherited metabolic disorders – and finally the objectives of the present work. Our studies initiated with the molecular characterization of all patients currently followed by the Portuguese metabolic centers (chapter 2). The rationale for this study was the previous description that GALT genotype represents a valuable prognostic tool for the outcome of galactosemic patients. Accordingly, after genotyping 42 Portuguese galactosemic patients, we searched for genotype-phenotype correlations by a 3-fold evaluation. Firstly, we employed in silico strategies to assess the structural-functional impact of previously uncharacterized mutations; secondly, we evaluated the biochemical phenotype at both the metabolite and enzymatic levels, represented by Gal-1-P values and GALT activity in erythrocytes; and finally, evaluated the resulting clinical outcome. Establishment of correlations between genotype and biochemical or clinical phenotypes, however, was poorly attained, reiterating the complexity of this disease and emphasizing the idea that other modifiers – possibly genetic, epigenetic and environmental – contribute to the pathophysiology of classic galactosemia. The update of the Portuguese GALT mutational spectrum revealed the intronic mutation c.820+13A>G (IVS8+13A>G) as the second most prevalent variation, strongly suggesting its pathogenicity, which set the basis for the study described in chapter 3. We functionally characterized the c.820+13A>G variation by ex vivo and in vivo analyses, which were in full agreement with the previous in silico predictions. Indeed, we confirmed this variation is a disease-causing mutation, whose mechanism of action involves the activation of a cryptic donor site, which, in turn, induces an aberrant splicing of the GALT pre-mRNA, thus causing a frameshift with inclusion of a premature stop codon. Structural-functional studies of the recombinant truncated GALT showed it was devoid of enzymatic activity and prone to aggregation. Finally, antisense oligonucleotides were designed to specifically recognize the mutation, and successfully restored the constitutive splicing. Molecular studies in several inherited metabolic disorders have led to the realization that only a minor part of the mutations directly disrupt functional sites of the proteins. Accordingly, in chapter 4, we described the structural and functional characterization of the most prevalent mutations in the GALT gene – p.Q188R, p.S135L, p.K285N and p.N314D – and of other five clinically relevant mutations – p.R148Q, p.G175D, p.P185S, p.R231C and p.R231H. Interestingly, the analyzed mutations did not affect the global conformational stability of the GALT enzyme; rather, most mutations, notwithstanding their impact on the enzyme functionality, increased the propensity for aggregation, which at the cellular level reflects in a decrease of the enzyme’s effective concentration. These results are in agreement with previous studies in classic galactosemia models, and suggest that GALT aggregation might be a major pathogenic mechanism underlying this disorder. Previous studies have reported a yeast galactosemia model allowing the evaluation of human GALT mutations severity, by assaying the sensitivity of transformed yeast cultures to galactose addition to the medium. Thus, in chapter 5, we developed a prokaryotic model of galactose sensitivity to evaluate the ability of the above referred human GALT mutants in alleviating the galactose-induced toxicity. This model presents the inherent advantage of being assayed in vivo, thus providing valuable insights on mutations’ impact on human GALT function. Additionally, arginine ability to ameliorate the galactose-induced toxicity was also evaluated for each human GALT mutant. The rationale for this approach was, not only the long-recognized anti-aggregation properties of arginine, but also its important therapeutic effect described in previous studies. In effect, arginine appears to exert a mutation-specific mode of action, alleviating the galactose toxicity in the p.Q188R, p.K285N, and p.G175D mutants, which suggests that might be of some benefit in classic galactosemia. Nonetheless, further studies are underway to ascertain arginine’s potential therapeutic effect in this inherited disorder of the galactose metabolism. Chapter 6 presents a general discussion and major conclusions disclosed by this work, framing them in the current state of the art, proving also some perspectives for future studies. Taken together, our results provide important insights on classic galactosemia, namely by shedding light on the underlying pathogenic molecular mechanisms, thus contributing for a better understanding of this enigmatic disorder. Finally, and importantly, these studies paved the way to the search, development and improvement of novel and alternative therapeutic strategies, so needed to overcome the overwhelming and burdensome long-term complications presented by most classic galactosemic patients.Após a sua internalização celular, a galactose é rapidamente metabolizada a glucose-1- fosfato pela acção sequencial de três enzimas: galactocinase (GALK), galactose-1-fosfato uridililtransferase (GALT) e uridina difosfogalactose 4’-epimerase (GALE). Estas enzimas permitem, respectivamente, a fosforilação da galactose em galactose-1-fosfato (GALK), a conversão de galactose-1-fosfato e uridina difosfoglucose em glucose-1-fosfato e uridina difosfogalactose (GALT), e a interconversão de uridina difosfoglucose em uridina difosfogalactose (GALE). Estas enzimas constituem a via de Leloir e, apesar de o fígado ser o principal órgão envolvido no metabolismo da galactose, encontram-se na maioria das células e dos tecidos. Uma deficiência enzimática em qualquer uma das três enzimas da via de Leloir resulta numa diminuição ou ausência de capacidade de metabolizar a galactose e, consequentemente, conduz à sua acumulação no sangue – hipergalactosémia. A Galactosémia Clássica (OMIM #230400), a forma mais comum de hipergalactosémia primária, é uma doença genética de transmissão autossómica recessiva, que afecta 1 em cada 30.000 a 60.000 nados-vivos, apresentando uma prevalência variável entre populações, nomeadamente na Irlanda e Turquia onde atinge valores de 1/23.500 - 1/23.775 e no Japão onde a sua prevalência atinge o valor mais baixo (1/1.000.000). A ausência ou diminuição de actividade da GALT é causada por mutações no gene GALT, e encontram-se descritas mais de 250 variações, reflectindo a elevada heterogeneidade alélica desta doença metabólica. Para além disso, a maioria dos doentes são heterozigóticos compostos, um dos factores determinantes da ampla variabilidade fenotípica observada. Aquando do nascimento, a criança aparenta ser assintomática. Os sintomas tornam-se evidentes após o início da ingestão de leite, e consistem inicialmente em dificuldades alimentares e de desenvolvimento, vómitos, diarreia, letargia e hipotonia, podendo evoluir para cataratas e septicémia, e eventualmente conduzir à morte. Após implementação de uma dieta restrita em galactose – o tratamento padrão – as crianças mostram notáveis melhorias em apenas 24 horas, e em apenas uma a duas semanas as disfunções hepática e renal desaparecem completamente. Esta resposta dramática à terapia dietética levou ao conceito da Galactosémia Clássica como uma doença relativamente benigna e fácil de tratar. No entanto, os doentes com Galactosémia Clássica desenvolvem complicações a longo prazo e a diversos níveis, como neurológico e psiconeurológico, crescimento e densidade óssea, e disfunção ovárica nas mulheres, os quais parecem ser independentes de um diagnóstico precoce e de uma adesão do doente à terapia. Apesar de a primeira descrição datar de 1908, a Galactosémia Clássica continua a ser um enigma, quer ao nível do conhecimento aprofundado da sua fisiopatologia, quer ao nível do desenvolvimento de alternativas terapêuticas que permitam, sobretudo, mitigar as complicações a longo prazo. O presente trabalho pretende, assim, contribuir para a elucidação dos mecanismos moleculares subjacentes à patogénese das mutações GALT prevalentes na população galactosémica Portuguesa, assim como explorar novas estratégias terapêuticas direccionadas para cada tipo específico de mutação. O primeiro capítulo da tese apresenta uma revisão geral da literatura sobre vários aspectos relacionados com a Galactosémia Clássica: as bases bioquímicas e moleculares, a fisiopatologia e as actuais abordagens terapêuticas. Em seguida, descrevem-se em pormenor os mecanismos moleculares subjacentes à maioria das mutações que originam Erros Hereditários do Metabolismo (onde se inclui a Galactosémia Clássica) e que causam perca de função, nomeadamente as mutações missense e as que afectam o splicing, mecanismos esses que condicionam as novas terapias desenhadas de acordo com o tipo de mutação. Por fim, são descritos os objectivos delineados para o presente trabalho. O trabalho experimental teve início com a caracterização molecular de todos os doentes actualmente seguidos nos centros metabólicos nacionais de Lisboa, Porto e Coimbra, sendo o objectivo final averiguar se, como sugerido na literatura, o genótipo GALT pode ter um valor prognóstico sobre a evolução fenotípica dos doentes galactosémicos (capítulo 2). Os dados obtidos permitiram estabelecer o espectro mutacional da Galactosémia Clássica em Portugal e compará-lo com o de outras populações. O estudo de 42 doentes revelou a presença de 14 substituições nucleotídicas, sendo 10 missense, 2 nonsense e 2 de splicing. Identificaram-se 16 genótipos diferentes, mas metade dos doentes são homozigóticos para p.Q188R, a mutação prevalente não só em Portugal como a nível mundial. Surpreendentemente, a segunda mutação mais frequente é uma mutação de splicing, descrita até então como benigna. Em seguida, e recorrendo a programas bioinformáticos adequados, procedeu-se à análise da potencial patogenicidade das mutações ainda não caracterizadas na literatura. Os resultados sugeriram que a maioria destas mutações missense afectará a estabilidade e a funcionalidade da proteína mutada, enquanto que a mutação de splicing mais frequente deverá induzir um mecanismo de splicing alternativo. Por fim, este estudo revelou, na maioria dos casos, a ausência de uma correlação clara entre a gravidade das mutações prevista pela análise in silico e o fenótipo bioquímico dos doentes, determinado pelos níveis eritrocitários de galactose-1-fosfato, assim como com o fenótipo clínico e a evolução desfavorável manifestada pelos doentes. No entanto, tal resultado não surpreende dado a Galactosémia Clássica, apesar de ser uma doença monogénica, não originar fenótipos claros. Este facto prende-se com a falta de informação estrutural sobre os componentes enzimáticos da via de Leloir, colocando-se a hipótese de uma alteração na GALT poder afectar toda a via metabólica. Por outro lado, os metabolitos da galactose estão envolvidos em diversas reacções fisiológicas, nomeadamente as de glicosilação que se reflectem aos mais variados níveis. Finalmente, a influência de outros genes modificadores, de alterações epigenéticas e de factores ambientais também não pode ser ignorada. Os capítulos seguintes são dedicados ao estudo dos mecanismos moleculares subjacentes às mutações prevalentes na população Portuguesa e ao desenvolvimento de novas abordagens terapêuticas. No capítulo 3 descreve-se o estudo da mutação de splicing IVS8+13A>G, até então considerada como benigna, mas que revelou ser a segunda mais frequente na nossa população galactosémica. A caracterização funcional do mecanismo patogénico foi efectuada por transfeção de um minigene contendo a sequência mutada em duas linhas diferentes de células eucarióticas. A análise dos produtos de transcrição revelou que a mutação activa um sítio críptico de splicing, causando um splicing anómalo do pré-mRNA GALT, o qual induz um frameshift com inclusão de um codão de terminação prematuro (p.D274GfsX291). Por outro lado, dado ter-se observado a presença do mensageiro mutado nas amostras biológicas dos doentes portadores desta mutação, colocou-se a hipótese de a proteína truncada ser produzida in vivo, o que levou à produção da proteína recombinante. Os estudos estruturais e funcionais subsequentes revelaram que esta proteína é propensa à formação de agregados e é destituída de actividade enzimática. Finalmente, e utilizando oligonucleótidos antisense que hibridam especificamente com o local da mutação, impedindo assim a ligação do spliceossoma e forçando a sua ligação ao sítio canónico dador de splicing, foi possível corrigir o splicing alternativo induzido pela mutação e obter o mensageiro selvagem. Estas experiências constituíram a prova de conceito sobre a aplicabilidade da terapia antisense como alternativa estratégica para a claramente insuficiente dieta restrita em galactose. ! O espectro mutacional da maioria dos erros hereditários do metabolismo é dominado por mutações do tipo missense. O capítulo 4 aborda a caracterização estrutural e funcional das quatro variantes GALT prevalentes a nível mundial, assim como outras cinco variantes com relevância clínica, nomeadamente na população galactosémica Portuguesa. Diversas metodologias foram empregues para determinação da actividade enzimática e do perfil de inactivação térmica, bem como métodos biofísicos para avaliação das estruturas secundária, terciária e quaternária das proteínas mutadas. Os resultados revelaram que as mutações pontuais não afectam nenhuma das estruturas acima mencionadas, mas sim a propensão para uma agregação precoce destas variantes. Este resultado constitui na realidade a principal novidade, dado que um estudo recente postulou que algumas mutações no gene GALT afectam o correcto folding das proteínas mutadas. Esta conclusão é extremamente relevante na medida em que, para além da diminuição da actividade uridililtransferásica, a acumulação de agregados proteicos interfere com a homeostase celular. Efectivamente, diversos estudos relataram a presença, em doentes galactosémicos, do aumento de actividade dos sistemas ligados ao stress do retículo endoplasmático e da unfolded protein response, assim como de níveis elevados de stress oxidativo, fenómenos característico de uma proteotoxicidade. Estes dados levam-nos então a colocar como potencial e nova hipótese terapêutica a utilização de moduladores da proteostase, os quais prolongam a semi-vida celular das variantes GALT, compensando assim parcialmente a diminuição de actividade enzimática e simultaneamente prevenindo a acumulação de agregados proteicos. No capítulo 5 desenvolvemos um modelo procariótico de sensibilidade à galactose que permitisse avaliar, não só o impacto causado pelas diversas mutações missense, mas também o efeito de compostos com potencial acção terapêutica. Utilizámos uma estirpe de E. coli com o gene galT endógeno deletado, de modo que toda a actividade GALT detectada é proveniente do mutante nela transformado. Os resultados, embora ainda preliminares, confirmaram a validade do modelo delineado e permitiram obter dados a dois níveis. Por um lado, replicámos in vivo os resultados previamente obtidos in vitro quanto à capacidade de aliviar a toxicidade induzida pela galactose, por comparação com a enzima selvagem. Por outro lado, e muito relevante, verificámos que o modelo é válido para testar moléculas com potencial terapêutico. Efectivamente, algumas destas enzimas mutadas revelaram-se sensíveis à arginina, um composto amplamente reconhecido como estabilizador de proteínas e anti-agregante, aliviando desse modo a toxicidade da galactose para a bactéria. A presente tese termina com o capítulo 6 no qual se apresenta uma discussão dos resultados obtidos, incluindo uma análise integrada de todo o trabalho e respectivas conclusões, bem como algumas perspectivas de trabalho futuro. Em suma, este trabalho contribuiu para um melhor conhecimento da Galactosémia Clássica em Portugal e para a elucidação dos mecanismos patogénicos subjacentes às mutações prevalentes, bem como para a descoberta e desenvolvimento de novas alternativas terapêuticas, tão necessárias para minorar as graves sequelas que a maioria dos doentes galactosémicos apresenta a longo termo.Fundação para a Ciência e a Tecnologia (FCT, SFRH/BD/48259/2008, projeto PEst-OE/SAU/UI4013/2011