Rozprawy doktorskie na temat „Modulating aggregation”

Alzheimer’s disease (AD) is a common, fatal polygenic neurodegenerative disorder, which still lacks satisfactory therapy or a preventative treatment. Among various hypotheses, the amyloid β (Aβ) cascade hypothesis is a widely accepted explanation by which Aβ aggregation is the causative process in AD pathology. Controlling this aggregation and understanding the structure of the resulting aggregates are core challenges to further understanding of AD. New, bioavailable small-molecule probes with programmable geometries, designed to interact with Aβ, have been obtained in high yields. These probes include functionalized azamacrocycles cyclam and cyclen, connected to pendants through triazole linkers that can coordinate to a central metal ion. It has been shown that probes with different structures have different abilities to influence Aβ aggregation, and therefore potentially capacity to protect neurons from Aβ toxicity by diverting the aggregation behavior of Aβ to alternative endpoints. The aim of this project is to synthesize new functionalized azamacrocycles, which can interact with Aβ and redirect aggregation pathways. A series of tetra-N-substituted cyclam compounds with various pendant groups and triazole group as the linker have been synthesized and characterized. The synthetic approach involves a nucleophilic substitution reaction, copper-catalyzed azide-alkyne cycloaddition (CuAAC) reactions, bromination, tetra-N-alkylation, protonation and metal complexation. Thioflavin T (ThT) fluorescence assays and small angle X-ray scattering (SAXS) experiments were conducted with the novel compounds on Aβ40/42 peptides and other amyloidogenic hydrophobins, the results of which will be discussed in terms of protein binding, surface tension modification, and fluorescence quenching

Dissertação para obtenção do Grau de Mestre em Genética Molecular e Biomedicina
It is widely known that α-synuclein (aSyn) is an amyloidogenic protein prone to aggregation. This protein is found in specific inclusions named Lewy bodies in the surviving neurons of Parkinsons’s disease patients and other synucleinopathy brains. This aggregation process is greatly affected by different post-translational modifications, such as phosphorylation, acetylation, and glycation. Lately it was shown that aSyn oligomeric species are more toxic than the inclusion bodies. Heat shock proteins (HSPs) are molecular chaperones able to modulate the folding and refolding of proteins. Its overexpression in Parkinson’s disease models reduces and prevents aSyn aggregation. As the reduction of aSyn aggregation can lead to an eventual accumulation of oligomeric species which may cause cell damage, the main goal of this work is to better understand the role of HSPs in aSyn oligomer formation, clarifying which are the aSyn resulting species formed in the presence of HSPs. Moreover, as glycation is suggested to accelerate abnormal protein deposition, we aimed to investigate how HSPs interfere with the oligomerization process of glycated aSyn. In this study Hsp70 seemed to induce recombinant aSyn oligomerization, generating higher molecular weight species with no associated toxicity. On the other hand, Hsp27 reduced aSyn oligomerization in vitro possibly by inducing the formation of non-reactive small oligomers. MGO glycation increased protein aggregation and cell death. Interestingly, Hsp27 overexpression reversed glycated aSyn aggregation and its associated toxicity. These results demonstrate the importance of HSPs modulation as a possible target of Parkinson’s disease therapeutics.

Aggregation of the amyloid-β (Aβ) peptide to form oligomers and amyloid fibrils is a central event in the pathogenesis of Alzheimer’s disease (AD). This thesis aims to provide a better understanding of Aβ toxicity and the impact of changes in Aβ aggregation, both relevant to AD. We have found that Aβ nitrotyrosination inhibits fibril formation, which favours the stabilization of small oligomers. We show that nitro-Aβ oligomers strongly bind to dendrites, altering N-methyl-D-aspartate receptor (NMDAR) physiological function and leading to neuronal dysfunction and cell death. Furthermore, we propose a model for Aβ fibril assembly, according to which fibril elongation is interrupted upon nitrotyrosination, due to the destabilization of interprotofibrillar contacts. Additionally, using a genome-wide screen in Saccharomyces cerevisiae, we have identified novel modulators of Aβ toxicity that are potential targets for the development of new AD therapeutic approaches.
L’agregació del pèptid b-amiloide (Aβ) en forma d’oligòmers i fibres és un esdeveniment central en la patogènesi de la malaltia d’Alzheimer. Aquesta tesi pretén aprofundir en els coneixements actuals sobre la toxicitat causada per l’Aβ així com en l’impacte que tenen els canvis en l’agregació d’aquest, tots dos rellevants per la malaltia d’Alzheimer. Els nostres resultats indiquen que la nitrotirosinació de l’Aβ inhibeix la formació de fibres, afavorint l’estabilització d’oligòmers. Demostrem que els oligòmers d’Aβ nitrat s’uneixen a les dendrites, alterant la funció fisiològica dels receptors d’N-metil- D-aspartat (NMDAR) i provocant disfuncions neuronals i la mort cel·lular. A més, proposem un model d’assemblatge per a les fibres d’Aβ, segons el qual la nitrotirosinació interromp l’elongació de la fibra a causa de la desestabilització dels contactes entre protofibres. Addicionalment, utilitzant un cribratge genòmic en Saccharomyces cerevisiae, hem identificat nous moduladors de la toxicitat causada per Aβ, que podrien ser clau per al desenvolupament de noves estratègies terapèutiques de la malaltia Alzheimer.

Tauopathien sind Krankheiten, die mit einer abnormen intrazellulären Tau-Protein-Faltung, Tau-Protein-Aggregation und Filament-Bildung im ZNS und PNS einhergehen. Die Alzheimer Demenz stellt die häufigste Tauopathie dar. Bei ihr kommt es zu einer intra- und extrazellulären Ablagerung fehlgefalteter amyloidogener Proteinaggregate, welche durch β Amyloid und Tau gebildet werden. Es resultiert eine Neurodegeneration. Ein multifaktorieller Prozess führt bei den Tauopathien zur Umwandlung des Tau-Proteins hin zu unlöslichen Fibrillenbündeln. Primär kommt es zur Bildung eines Dimers, das durch Disulfid- und Wasserstoffbrückenbindungen stabilisiert wird. Durch die Zusammenlagerung mehrer Dimere entstehen Tau-Oligomere. Diese gelten als die neurotoxischen Komponenten. Die Primärstruktur des Tau-Proteins beeinflusst den Aggregationsprozess. Dabei spielen die Anwesenheit der Aggregationsdomänen PHF6* und PHF6 und die Anzahl der Cystein-Seitenketten überragende Rollen. In dieser Arbeit wurden durch ortsspezifische Mutagenesen Tau-Konstrukte generiert, die sich in ihrer Anzahl der Aggregationsdomänen und der Cystein-Reste unterschieden. Es konnte gezeigt werden, dass die Aggregationstendenz der Tau-Proteine mit nur einer Aggregationsdomäne und keinem Sulfhydryl-Rest stark sinkt. Auch durch Veränderungen des Redoxmilieus lässt sich das Tau-Aggregationsverhalten beeinflussen. Es wurde gezeigt, dass die Substanz TCEP als starkes Reduktionsmittel die Aggregation der 2 humanen Tau-Isoformen 2N3R und 2N4R wirkungsvoll inhibiert. Auch GSSG als Oxidationsmittel verhinderte in bestimmten Konzentrationen die Aggregation dieser 2 Isoformen. Es wird angenommen, dass die Verhinderung der Bildung einer intermolekularen Disulfidbrückenbindung zu diesem Phänomen führt.

Mutations in the E3 ubiquitin ligase Parkin are associated with autosomal recessive juvenile Parkinsonism. Furthermore, Parkin is recruited to Lewy bodies in brains of patients with sporadic Parkinson’s disease (PD), suggesting a role for Parkin in the pathogenesis of PD. This study investigated the effect of the DnaJ/Hsp40-like protein HSJ1 (DnaJB2) on Parkin biology. HSJ1 proteins are neuronal cochaperones that regulate Hsp70 activity through their J domain and exploit ubiquitin interaction motifs (UIM) to promote sorting of misfolded ubiquitylated proteins to the proteasome for degradation. Disease-related mutant forms of Parkin formed inclusions when overexpressed in neuronal cells. Co-expression of mutant Parkin with HSJ1a significantly reduced inclusion formation and altered inclusion characteristics. The effect of HSJ1a on Parkin inclusions required a functional J domain. Mutant Parkin could be co-immunoprecipitated with HSJ1a. Wild-type Parkin was also co-immunoprecipitated with HSJ1a, suggesting a potential interaction with normal Parkin. Importantly, HSJ1a enhanced the E3 ubiquitin ligase activity of wild-type Parkin, dependent on the HSJ1 UIM domain. HSJ1 and Parkin promoted the degradation of the Parkin substrate Synphilin-1. Finally, it was confirmed that Parkin relocates to mitochondria upon mitochondrial membrane depolarization. Moreover, Parkin induced the selective elimination of impaired mitochondria by mitophagy. Interestingly, HSJ1 rescued the relocation of Parkin mutants to damaged mitochondria and partially restored mutant Parkin’s ability to induce mitophagy. These data show that HSJ1 proteins may affect Parkin aggregation and function, which may have a broader relevance to PD.

Neurofibrillary tangles (NFT) in Alzheimer's disease (AD) are mainly composed of hyperphosphorylated and aggregated wild-type tau. NFTs are decorated by the ubiquitin-like modifier NEDD8, a protein targeted for proteasomal degradation by the NEDD8 Ultimate Buster 1 (NUB1). NUB1 has been shown to reduce synphilin-1 positive inclusions in a model of Parkinson’s disease. Therefore, this study examined the subcellular localisation of NUB1 as well as the effect of NUB1 on tau phosphorylation and aggregation. Furthermore, the effect of reducing NUB1 expression by RNA interference was investigated. Brain sections from AD patients showed that NUB1 and NEDD8 were expressed in the pyramidal neurons of the hippocampus, where the accumulation of NFTs is most abundant. In rat primary cortical neurons, NUB1 and tau co-localised in neurites and signalling structures such as varicosities, suggesting a functional interaction between them. The upregulation of the tau kinase GSK3β in AD leads to increased tau hyperphosphorylation and accumulation. In SK-N-SH neuroblastoma cells, which lack endogenous tau, ectopic wild-type tau formed inclusions when it was co-expressed with GSK3β, and this was enhanced by proteasome inhibition. NUB1 co-localised with both tau and GSK3β and significantly reduced tau inclusion formation. In neuroblastoma cells, NUB1 could interact with both tau and GSK3β, disrupt their interaction, and decrease the GSK3β-dependent phosphorylation of tau. NUB1 can directly bind synphilin-1 and induce its proteasomal degradation. Therefore, the ability of NUB1 to regulate GSK3β degradation was investigated in neuroblastoma cells. The upregulation of NUB1 accelerated the turnover of GSK3β, and the ubiquitin-associated (UBA) domains of NUB1 were necessary for NUB1 to exert its effect. Conversely, the downregulation of endogenous NUB1 by RNA interference increased the stability of endogenous GSK3β. Thus, NUB1 might have a role in tau inclusion formation by modulating GSK3β levels.

La maladie d'Alzheimer est la démence la plus répandue dans le monde. Le nombre de cas augmente de manière exponentielle et il est donc important de comprendre les mécanismes moléculaires donnant lieu à cette terrible maladie. Une des hypothèses les plus supportées est celle suggérant que la production et dégradation déséquilibrées de l'amyloïde-beta (Aß), un peptide de 42 acides aminés trouvé dans tous les individus sains, est un événement clé dans le déroulement de la maladie d'Alzheimer. En effet, une production accrue ou une dégradation faible du peptide ont pour conséquence son agrégation et accumulation dans des plaques de fibres entre les neurones des régions spécifiques du cerveau. C'est pourquoi la modulation de l'agrégation du peptide Aß est une des approches envisageables pour modifier l'évolution de la maladie d'Alzheimer. Les protéines chaperons dont une des fonctions est d'assister d'autres protéines dans leur repliement, sont parmi les molécules les plus étudiées pour leur capacité modulatrice de l'agrégation des protéines (inclus le peptide Aß). Plusieurs chaperons ont montré la capacité d'inhiber la formation des fibres par l'Aß. Cependant, du fait que les chaperons sont des molécules conservées et peu spécifiques, leur surexpression ou administration directe peut avoir des conséquences négatives si les chaperons interagissent avec des protéines autres que la protéine cible. Dans ce travail, nous nous sommes intéressés à une protéine chaperon bactérienne possédant une forte activité " holdase " (i.e., elle empêche le repliement précoce des protéines) comme possible modulateur de l'agrégation du peptide Aß. Le chaperon sauvage a une très faible capacité d'inhibition de la formation de fibres par le peptide Aß. Cependant, nous avons démontré qu'en modifiant légèrement la surface de liaison du chaperon, la protéine devient un puissant inhibiteur de l'agrégation d'Aß. En parallèle, nous nous sommes intéressés à l'influence des ions métalliques sur l'agrégation du peptide Aß. [...]
Alzheimer's disease is the most frequent type of dementia. With an exponentially growing number of cases, understanding the underlying molecular events leading to this devastating condition is of crucial importance. Much evidence points to a disequilibrium in the production and degradation of amyloid beta (Aß), a normally physiological 42 amino acid peptide, as an early key event in Alzheimer's etiology. Whether Aß is overproduced or poorly degraded, the overall result is an abnormally large pool of peptide that gradually aggregates forming extracellular deposits of fibrils, called amyloid plaques, in specific brain regions. Hence, modulation of Aß aggregation process is one of the suggested approaches to control the evolution of Alzheimer's disease. Universally conserved molecular chaperones have been intensively studied for their capacity to prevent aggregation of disease-related proteins, and many of them have proven to efficiently modulate Alzheimer's Aß aggregation. In a scenario where chaperones are overexpressed or directly administered into the affected tissue, the universal conservation and the relatively poor client-specificity of generic chaperones can become a downside because of the risk of interaction with proteins other than the targeted one is not dismissible, and thus the consequences unpredictable. In the first part of this work, we looked upon a bacterial chaperone call SecB with an unusually robust holdase activity (i.e. it prevents early protein folding) as a promising modulator of Alzheimer's Aß peptide aggregation. [...]

Misfolding of proteins and peptides is closely linked to several neurodegenerative disorders, among them Alzheimer's disease (AD), the most prominent example of brain diseases. The self-assembly of the amyloid β peptide (Aβ) into amyloid fibrils is one histologic hallmark of AD. A detailed knowledge about the underlying mechanism(s) of Aβ aggregation is crucial for advances toward a fundamental understanding of the disease, which may promote the search for and design of efficient therapeutics. The work presented in this thesis deals with modulation of the aggregation process by various compounds, i.e. small organic molecules (e.g. lacmoid and Congo red), surfactants and metal ions. These results provide insight into the molecular mechanism of modulator interactions and interference with Aβ and its aggregation pathways. Applying a combination of kinetic and dynamic studies as well as structural investigations we characterized the molecular interactions between Aβ and aggregation modulators in terms of microscopic rate constants, conformational preferences and thermodynamics. An important conclusion is that these modulators form highly dynamic complexes with Aβ, with life-times on the timescale of milliseconds. Despite the similar exchange dynamics, the effect on peptide aggregation is modulator-specific and fibril formation can be accelerated, retarded or inhibited by their interactions. In summary, Aβ self-assembly is governed by microscopic kinetic and dynamic processes that can be altered by aggregation modulators. Further elucidation of these mechanisms is beneficial for the understanding and therapeutic intervention of amyloid diseases.

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.

De nombreuses études ont mis en évidence l’importance du contrôle qualité des protéines, c’est à dire des mécanismes de reconformation (chaperons moléculaires) et de dégradation (autophagie, proteasome) des protéines dans différentes pathologies musculaires telles que la dystrophie musculaire d’Ullrich (UCMD), de Duchenne (DMD) ou d’Emery-Dreifuss (EDMD) ; cependant, à l’heure actuelle, aucune n’a été menée sur l’ensemble de ces mécanismes dans un seul et même modèle et sur des cellules musculaires avant leur différenciation en muscles. Nous nous sommes donc intéressés à la fonctionnalité des mécanismes de Contrôle Qualité des Protéines et à leurs interconnexions dans des myoblastes immortalisés de donneurs sains ou de patients atteints de DMD. Nous avons observé une augmentation de l’agrégation protéique dans les cellules DMD. Ce phénomène s’accompagne d’une dérégulation des mécanismes de séquestration par les chaperons moléculaires, conséquence d’une modulation de l’expression des protéines HSPB5 et HSPB8. Les mécanismes de dégradation sont également dérégulés; en effet, nous avons observé d’une part, une diminution de l’activité enzymatique du protéasome ainsi que des molécules d’adressage des protéines multiubiquitinées au protéasome et d’autre part, une augmentation de l’activité du facteur de transcription NF?B, de l’expression de protéines intervenant dans l’autophagie et des complexes BAG3/HspB8 conduisant à une augmentation du flux autophagique. L’ensemble de ces dérégulations reflète l’existence d’un stress d’agrégation protéique dans les myoblastes issus de patients DMD. Dans ce contexte, la modulation pharmacologique du PQC dans ces cellules pourrait représenter une nouvelle stratégie thérapeutique pour la Dystrophie Musculaire de Duchenne
Various studies have highlighted the importance of Protein Quality Control (PQC), including protein refolding (molecular chaperones) and degradation (autophagy, proteasome) mechanisms in inherited muscle disorders such as Ullrich Congenital Muscular Dystrophy (UCMD), Duchenne Muscular Dystrophy (DMD) or Emery-Dreifuss Muscular Dystrophy (EDMD); however, to date, no extensive study has been conducted on these mechanisms in a same model, in muscle cells before muscle differentiation. Thus, we were interested in PQC mechanisms functionality and their interconnection in human immortalized myoblasts from healthy donors or patients suffering from DMD. We observed an increase of protein aggregation in DMD cells. This phenomenon is accompanied by a deregulation of sequestration mechanisms by molecular chaperones, reflected by the modulation of HSPB5 and HSPB8 expression. Degradation mechanisms are also deregulated; indeed, we observed on one hand a decrease of proteasome enzymatic activity and multiubiquitinated proteins UPS-adressing molecules and on the other hand, an increase of NF?B transcription factor’s activity, involved in autophagy, and of BAG3/HSPB8 complexes, leading to an increase of the autophagic flux. These PQC defects reflect the existence of a protein aggregation stress in myoblasts coming from DMD patients. In this context, pharmacological modulation of PQC in these cells could represent a new therapeutic strategy for Duchenne Muscular Dystrophy

"Topographical and mechanical properties of adhesive substrates provide important biological cues that affect cell spreading, migration, growth, and differentiation. The phenomenon has led to the increased use of topographically patterned and flexible substrates in studying cultured cells. However, these studies may be complicated by various limitations. For example, the effects of ligand distribution and porosity are affected by topographical features of 3D biological constructs. Similarly, many studies of mechanical cues are compounded with cellular deformation from external forces, or limited by comparative studies of separate cells on different substrates. Furthermore, understanding cell responses to mechanical input is dependent upon reliable measurements of mechanical properties. This work addresses each of these issues. To determine how substrate topography and focal adhesion kinase (FAK) affect cell shape and movement, I studied FAK-null (FAK -/-) and wild type mouse 3T3 fibroblasts on chemically identical polystyrene substrates with either flat surfaces or micron-sized pillars, I found that, compared to cells on flat surfaces, those on pillar substrates showed a more branched shape, an increased linear speed, and a decreased directional stability, which were dependent on both myosin-II and FAK. To study the dynamic responses to changes in substrate stiffness without other confounding effects, I developed a UV-modulatable substrate that softens upon UV irradiation. As atomic force microscopy (AFM) proved inadequate to detect microscale changes in stiffness, I first developed and validated a microsphere indentation method that is compatible with fluorescence microscopy. The results obtained with this method were comparable to those obtained with AFM. The UV-modulatable substrates softened by ~20-30% with an intensity of irradiation that has no detectable effect on 3T3 cells on control surfaces. Cells responded to global softening of the substrate with an initial retraction followed by a gradual reduction in spread area. Precise spatial control of softening is also possible - while there was little response to posterior softening, anterior softening elicited a pronounced retraction and either a reversal of cell polarity or a significant decrease in spread area if the cells move into the softened region. In conclusion, these techniques provide advances in gaining mechanistic insight into cellular responses to topographical and mechanical cues. Additionally, there are various other potential applications of the novel UV-softening substrate, particularly in regenerative medicine and tissue engineering. "

Protein aggregation is induced by a wide variety of cellular stresses, including amino acid starvation, virus infection, endoplasmic reticulum stress, lipopolysaccharide, and oxidative stress. It has been suggested that altered proteostasis impacts the inflammatory response, but the underlying mechanism between altered proteostasis and inflammation is still poorly understood. Here, we aim to analyse the impact of protein aggregation in the response of human Dendritic Cells, focusing on plasmacytoid dendritic cells (pDCs). The number of aggregates was manipulated in a pDC cell line, CAL-1, by using both autophagy and proteasome inhibitors. Proteasome inhibition in pDCs induced assembly of large p62-based aggregates, together with an increase on IL-1β secretion and cell death, in an irreversible and cell specific manner. The same effects were not observed upon autophagy inhibition nor on a monocytic cell line. To study the mechanism behind the increase in the inflammatory response upon proteasome inhibition, p62-knockout cells were generated using the CRISPR/Cas9 system. Our data suggests that neither p62 nor the NLRP3 inflammasome are required for induction of cell death upon proteasome inhibition in pDCs. Overall, we conclude that proteasome inhibition induces an inflammatory response specific to pDCs. We propose that this effect must be considered when using proteasome inhibitors as potential drugs for the treatment of pDCs derived immune-mediated disorder and thus, mores studies should be done to clarify the outcome of proteasome inhibition on pDCs.
A agregação de proteínas é um processo que pode ser induzido por uma grande variedade de stresses celulares, incluindo privação de aminoácidos, infeção viral, stress do retículo endoplasmático, lipopolissacarídeos, e stress oxidativo. Alterações na proteostase parecem ter um impacto na resposta inflamatória, mas os mecanismos subjacentes a este impacto são ainda pouco conhecidos. Esta tese tem por objetivo analisar de que forma a agregação de proteínas influencia a resposta das células dendríticas humanas, em particular das células dendríticas plasmocitóides (pDCs). Foram utilizados inibidores de autofagia e do proteossoma para manipular o número de agregados na linha celular de pDCs, CAL-1. A inibição do proteossoma em pDCs induziu a formação de agregados constituídos por p62 de grandes dimensões, e levou à secreção de IL-1β e morte celular, de forma irreversível e específica destas células. Os mesmos efeitos não foram observados após inibição da autofagia nem se verificaram numa outra linha celular, monocítica. Para estudar o mecanismo por detrás do aumento da resposta inflamatória após a inibição do proteossoma, foram criadas células knockout para o p62 usando o sistema CRISPR/Cas9. Os nossos resultados sugerem que nem o p62 nem o inflamassoma de NLRP3 são necessários para a indução da morte celular por inibição do proteossoma em pDCs. De forma geral, concluímos que a inibição do proteossoma induz uma resposta inflamatória específica para pDCs. Propomos que esse efeito deve ser tido em consideração quando se utilizem inibidores de proteossoma como potenciais fármacos para o tratamento de distúrbios mediados por pDCs e, portanto, mais estudos devem ser feitos para esclarecer o efeito da inibição do proteossoma em pDCs.
Mestrado em Biomedicina Molecular

Tauopathien sind Krankheiten, die mit einer abnormen intrazellulären Tau-Protein-Faltung, Tau-Protein-Aggregation und Filament-Bildung im ZNS und PNS einhergehen. Die Alzheimer Demenz stellt die häufigste Tauopathie dar. Bei ihr kommt es zu einer intra- und extrazellulären Ablagerung fehlgefalteter amyloidogener Proteinaggregate, welche durch β Amyloid und Tau gebildet werden. Es resultiert eine Neurodegeneration. Ein multifaktorieller Prozess führt bei den Tauopathien zur Umwandlung des Tau-Proteins hin zu unlöslichen Fibrillenbündeln. Primär kommt es zur Bildung eines Dimers, das durch Disulfid- und Wasserstoffbrückenbindungen stabilisiert wird. Durch die Zusammenlagerung mehrer Dimere entstehen Tau-Oligomere. Diese gelten als die neurotoxischen Komponenten. Die Primärstruktur des Tau-Proteins beeinflusst den Aggregationsprozess. Dabei spielen die Anwesenheit der Aggregationsdomänen PHF6* und PHF6 und die Anzahl der Cystein-Seitenketten überragende Rollen. In dieser Arbeit wurden durch ortsspezifische Mutagenesen Tau-Konstrukte generiert, die sich in ihrer Anzahl der Aggregationsdomänen und der Cystein-Reste unterschieden. Es konnte gezeigt werden, dass die Aggregationstendenz der Tau-Proteine mit nur einer Aggregationsdomäne und keinem Sulfhydryl-Rest stark sinkt. Auch durch Veränderungen des Redoxmilieus lässt sich das Tau-Aggregationsverhalten beeinflussen. Es wurde gezeigt, dass die Substanz TCEP als starkes Reduktionsmittel die Aggregation der 2 humanen Tau-Isoformen 2N3R und 2N4R wirkungsvoll inhibiert. Auch GSSG als Oxidationsmittel verhinderte in bestimmten Konzentrationen die Aggregation dieser 2 Isoformen. Es wird angenommen, dass die Verhinderung der Bildung einer intermolekularen Disulfidbrückenbindung zu diesem Phänomen führt.

Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2008.
Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3651. Adviser: Michael S. Strano. Includes bibliographical references. Available on microfilm from Pro Quest Information and Learning.

Tese de mestrado, Biologia Molecular e Genética, Universidade de Lisboa, Faculdade de Ciências, 2020
Phenylketonuria (PKU; OMIM #261600) is an autosomal recessive disorder, caused mostly by missense mutation in the PAH gene, which encodes human phenylalanine hydroxylase (hPAH).Human PAH is a nonheme, homotetrameric, iron-containing enzyme, which catalyzes the conversion of LPhenylalanine (L-Phe) into L-Tyrosine, by para-hydroxylation of the aromatic side chain in the presence of the co-factor (6R)-L-erytro-5,6,7,8-tetrahydrobiopterin (BH4), non-hemic iron and molecular oxygen (O2). Presently, classical PKU patients must follow restricted diets in order to control L-Phe levels in the blood. Compliance is difficult and cognitive and social development impaired. PKU can be classified as a misfolding disease, since the majority of the PKU causing mutations result in misfolded hPAH variants which, in the cellular context, will form soluble aggregates targeted for degradation, leading to a loss-of-function phenotype. Therefore, pharmacological chaperone therapy imposes a viable alternative to PKU treatment. By stabilizing the variant protein, and avoiding its degradation, rescue of enzyme residual activity may be attained, improving patient’s quality of life by increasing dietary LPhe tolerance and alleviating some of the dietary restrictions imposed. Among the high number of PAH variants already identify, the p. G46S is considered an excellent model to the in vitro study of the hPAH aggregation process, as it presents a high tendency to selfassociate and form non-amyloid fibrils, when overexpressed in E.coli, and to be rapidly degraded in eukaryotic systems, representing the most severe form of Classical PKU phenotype. In the presence of maltose binding protein (MBP) added N-terminally to p.G46S, it is possible to obtain soluble metastable tetramers with a normal enzyme activity rate. Upon MBP tag cleavage with Factor Xa protease the hPAH p.G46S aggregates in a process efficiently followed in vitro. Our aim was to identify, among an in-house compound library, molecules able to modulate this behavior in vitro, and to characterize the underlying mechanism by studying the full-length (FL) and a truncated form of p.G46S representing the unstable N-terminal regulatory domain (N1-120) harboring the allosteric site. For this, the p.G46S forms were expressed in a prokaryotic system in fusion with the MBP tag and purified. The rate of self-association of the isolated FL tetramers and N1-120 dimers (variant and wild-type) were studied by real-time turbidimetry, upon cleavage of the MBP tag, in the presence and absence of studied molecules. Following in vitro studies, molecules with a positive impact on p.G46S aggregation were submitted to in cellullo studies to evaluate their influence on enzyme activity and stability. From the aggregation assays, five molecules had positive effects on the self-association pattern of the FL p.G46S, either by inhibiting aggregation or by delaying it. From those molecules, two (C4 and C14) also inhibited the wild-type and p.G46S RD1-120 aggregation, and one (C17) only impacted the aggregation of the wild-type RD1-120, suggesting that these molecules bind to the unstable N-terminal domain and particularly C4 and C14 will be interacting with helix a1 of the ACT domain, the main driver of p.G46S-RD1-120 instability while C17 is acting on a different region. In cellullo assays showed that C17 and C14 could improve enzyme activity when compared to p.G46S in the absence of compounds (control). Enzyme content in cell lysates, however, was at the same level as the p.G46S control, thus suggesting that these two compounds are promoting a stabilization of the enzymatic reaction, allowing it to present higher activity levels than it normally would have (activity chaperone), instead of acting as stabilizers of the protein helping it to avoid the cell protein control quality mechanisms (pharmacological chaperone). Although further studies were not carried out due to the restrictions and contingency plans applied by the Research institutions to respond to SARS-CoV-2_COVID-19 pandemics, here we present possible hit structures for the development of a new class of pharmacological/activity chaperones, specifically designed for the treatment of classical PKU.
A fenilcetonuria (PKU; OMIM #261600) e uma doenca autossomica recessiva, causada na sua maioria por mutacoes missense no gene PAH, que codifica a fenilalanina hidroxilase humana (hPAH). A hPAH e uma enzima homotetramerica, que catalisa a conversao da L-fenilalanina (L-Phe) em Ltirosina (L-Tyr), por para-hidroxilacao da cadeia lateral aromatica. Para que esta reacao ocorra, e necessaria a presenca do seu co-factor natural (6R)-L-erytro-5,6,7,8-tetrahidrobiopterina (BH4), de ferro nao-hemico e oxigenio molecular (O2). As mutacoes missense no gene PAH originam na sua maioria a expressao de variantes de hPAH conformacionalmente alteradas (misfolded), no contexto celular, tem tendencia a formar agregados soluveis que rapidamente se tornam alvos das vias celulares de degradação proteica, levando a um fenotipo patologico de perda de funcao. A PKU e entao considerada uma doença conformacional, na qual o equilibrio normal dos estados folded« unfolded e alterado, deslocando-se preferencialmente para o estado unfolded. Consequentemente, as proteinas resultantes misfolded são reconhecidas, levando a que a maquinaria celular de controlo de qualidade e degradacao proteica atue no sentido de acelerar a degradacao de proteinas misfolded devido a sua instabilidade e tendencia para agregacao. Nos doentes PKU, quando nao tratados o mais rapidamente possivel apos diagnostico, a função alterada da hPAH variante resulta na acumulacao de L-Phe para niveis neurotoxicos, causando alteracoes neurologicas, convulsoes, atraso no desenvolvimento, problemas comportamentais, etc. O tratamento disponivel assenta basicamente em dietas restritivas e suplementos alimentares desde o diagnostico (preferencialmente dentro dos primeiros dias de vida) a fim de controlar os niveis de L-Phe em circulacao no sangue. Atualmente, as guidelines internacionais indicam que a restricao dietetica deve ser mantida durante a vida e que os niveis de L-Phe circulante devem ser mantidos abaixo de 600 μM (considerando niveis normais de circulacao no sangue ≈120 μM). A aquiescencia para com esta terapeutica e dificil, principalmente durante a adolescencia e nos adultos, e o desenvolvimento cognitivo e social sao prejudicados. Ao longo dos anos tem-se vindo a estudar diferentes terapeuticas alternativas, de forma a que seja possivel controlar os niveis de L-Phe circulantes e seja possivel um alivio nas restricoes dieteticas. A terapeutica baseada na administracao de chaperones propoem-se como uma alternativa viavel, uma vez que a PKU e atualmente reconhecida como uma doenca conformacional. Ao estabilizar a enzima misfolded e a sua degradacao iminente, permite-se que a enzima esteja presente e mantenha a sua atividade residual. Um leve aumento da capacidade de processamento da L-Phe poderá entao levar a um aumento da qualidade de vida do doente, aliviando algumas das restricoes dietéticas impostas. Ate a data, foram descritas tres abordagens diferentes para a terapia por chaperones: modulacao de chaperones moleculares a fim de resgatar proteinas com folding incorreto (reguladores de proteostase); chaperones quimicos e chaperones farmacologicos com o objetivo de estabilizar a proteina alterada, evitando-se a degradacao pelos sistemas de controlo de qualidade proteica. Entre as varias variantes causadoras de PKU, a p.G46S e considerada como um excelente modelo para o estudo in vitro do processo de agregacao da hPAH, uma vez que apresenta uma elevada tendencia para se autoassociar e formar fibrilhas nao amiloides, quando sobre-expressa em E.coli, e para ser rapidamente degradada em sistemas eucariotas, representando um fenotipo classico de PKU. Assim, e na presença da proteina de ligacao a maltose (maltose binding protein - MBP) adicionada a N-terminal da cadeia polipeptidica da p.G46S, e possivel obter tetrameros soluveis, metastaveis e com uma taxa de atividade enzimatica normal. Apos a clivagem da MBP com a protease Factor Xa (FXa), e entao possivel seguir in vitro e de forma eficiente o processo de agregacao da proteina hPAH p.G46S. Tendo em consideracao as caracteristicas descritas da p.G46S, o objetivo do presente estudo e o de identificar, de entre uma biblioteca de compostos desenhados e sintetizados pelo Grupo de Quimica Bioorganica do Instituto de Investigacao do Medicamento (iMed.ULisboa), moleculas capazes de modular o comportamento auto-associativo in vitro, e caracterizar o mecanismo subjacente atraves do estudo da proteina inteira (Full lenght; FL) e de uma forma truncada da p.G46S representando o domínio regulador instavel N-terminal (RD1-120) da hPAH e que contem o local de ligacao alosterico ao substrato L-Phe . Para tal, as formas de p.G46S (FL e RD1-120) foram expressas num sistema procariota como proteinas de fusao com a MBP e posteriormente purificadas. A taxa de auto-associacao dos tetrâmeros FL isolados e dos dimeros RD1-120 foram estudados por turbidimetria em tempo real, apos a clivagem da MBP, na presenca (100 μM) e ausencia das moleculas estudadas. Apos estudos in vitro, as moléculas com efeito inibidor na agregacao da p.G46S foram sujeitas a estudos in celullo com a finalidade de avaliar a sua influencia na atividade e estabilidade enzimatica na celula. Desta forma, células embrionicas do rim humano (human embryonic kidney cells), HEK293T, foram transfetadas com vetores de expressao eucariotas capazes de expressar a proteina p.G46S. Como controlo do ensaio celulas HEK293T foram tambem transfetadas com vetor de expressao contendo o gene normal da hPAH (wild type; WT). Para monitorizacao da estabilidade e variacao na tendencia de agregacao da proteína nas celulas eucariotas, foram realizados ensaios de imunocitoquimica. Adicionalmente, avaliou-se de que forma a funcao biologica celular da p.G46S seria afetada pela presenca dos compostos atraves da medicao da atividade enzimatica em lisados celulares. Dos ensaios de agregacao observou-se que cinco moleculas (C4, C10, C12, C14 e C17) tiveram efeitos positivos sobre o padrao de auto-associacao da forma FL da p.G46S, quer inibindo a agregacao, quer atrasando-a. Destas moleculas, duas (C4 e C14) atrasaram tambem a agregacao das formas truncadas representantes do RD (p.G46S e WT), enquanto uma (C17) apenas diminuia a agregacao da forma WT RD1-120, sugerindo que a interacao destas moleculas com a enzima ocorrera no dominio regulador N-terminal e que em particular as moléculas C4 e C14 deverao interagir com a helice a1 do dominio ACT promotora da instabilidade proteica. Estas cinco moleculas foram submetidas a estudos de imunocitoquimica, que, infelizmente, foram pouco conclusivos, devido a sobre-expressao tanto da proteina p.G46S como da WT, que impediu a discriminacao de agregados proteicos na celula. Desta forma, nao foi possivel obter resultados conclusivos acerca da capacidade de os compostos modularem a agregacao da proteina in cellullo. Para a avaliacao da modulacao da atividade biologica da proteina na celula pelos compostos em estudo (50 μM), realizaram-se ensaios na presenca de 0,5% DMSO e de 50 μM de C6 como controlos negativos. Como esperado, em ambos os ensaios a atividade enzimatica foi reduzida (» 15%), quando comparada com a hPAH WT. Estes resultados encontram-se em concordancia com os dados obtidos por analise de Western blot, sendo que os niveis intracelulares da proteina alvo se encontravam tambem reduzidos, quando comparados com a WT. Dos compostos que melhoravam a agregacao de p.G46S in vitro, tanto o C14 como o C17 mostraram ser capazes de melhorar a atividade da proteina variante, quando comparado com o ensaio controlo (p.G46S/0,5% DMSO). No entanto, para corroborar os pressupostos assumidos nas experiencias in vitro, ensaios adicionais deverao ser realizados, uma vez que se verificaram diferencas significativas entre os ensaios de atividade independentes, efetuados nas células HEK293T. Ao contrario do que seria de esperar, observou-se nos immunoblots, tanto para C14 como para C17, uma quantidade de proteina p.G46S tambem reduzida em comparacao com a hPAH WT, sugerindo que estes compostos poderao estar a estabilizar o processo enzimatico de modo a que esta tenha maior capacidade para realizar a sua funcao biologica (chaperone de atividade), ao inves de estabilizar a conformacao proteica, evitando a sua degradacao pela maquinaria de controlo de qualidade e degradacao proteica celular (chaperone farmacologico). Embora nao tenha sido possivel efetuar ensaios adicionais devido as restricoes impostas pelo plano de contingencia de combate a pandemia SARS-CoV-2_COVID-19, o presente estudo serviu como base para a identificacao de estruturas hit para o desenvolvimento de uma nova classe de chaperones farmacologicos ou chaperones de atividade direcionados para tratamento da PKU classica.