Dissertations / Theses on the topic 'Post-Translational Protein Processing'

Innate immunity is the first line of defense against invading pathogens. It provides immediate protection by initiating both cellular and humoral immune reactions in response to a wide range of infections. It is also important to the development of long-lasting and pathogen-specific adaptive immunity. Thus, studying of the innate immunity, especially the pathogen recognition and signaling modulation, is crucial for understanding the intrinsic mechanisms underlying the host defense, as well as contributing the development of the fight against infectious diseases. Drosophila is an ideal model organism for study of innate immunity. Comparing to mammals, Drosophila immunity is relative conserved and less redundant. A variety of molecular and genetic tools available add further convenience to the research in this system. My work is focused on the signaling modulation by post-translational modification after activation. In these studies I demonstrated in the center of Imd pathway, the Imd protein undergoes proteolytic cleavage, K63-polyubiquitination, phosphorylation, K63-deubiquitination and K48-polyubiquitination/degradation in a stimulation-dependent manner. These modifications of Imd play a crucial role in regulating signaling in response to infection. The characterization of ubiquitin-editing event provides a new insight into the molecular mechanisms underlying the activation and termination of insect immune signaling pathway.

Innate immunity is the first line of defense against invading pathogens. It provides immediate protection by initiating both cellular and humoral immune reactions in response to a wide range of infections. It is also important to the development of long-lasting and pathogen-specific adaptive immunity. Thus, studying of the innate immunity, especially the pathogen recognition and signaling modulation, is crucial for understanding the intrinsic mechanisms underlying the host defense, as well as contributing the development of the fight against infectious diseases. Drosophila is an ideal model organism for study of innate immunity. Comparing to mammals, Drosophila immunity is relative conserved and less redundant. A variety of molecular and genetic tools available add further convenience to the research in this system. My work is focused on the signaling modulation by post-translational modification after activation. In these studies I demonstrated in the center of Imd pathway, the Imd protein undergoes proteolytic cleavage, K63-polyubiquitination, phosphorylation, K63-deubiquitination and K48-polyubiquitination/degradation in a stimulation-dependent manner. These modifications of Imd play a crucial role in regulating signaling in response to infection. The characterization of ubiquitin-editing event provides a new insight into the molecular mechanisms underlying the activation and termination of insect immune signaling pathway.

The lectin Concanavalin A is processed in planta by a splicing-mediated circular permutation of its initial single-chain precursor, pro-Concanavalin A. Active (carbohydrate-binding) protein conformations can be purified by dextran-affinity chromatography. This thesis demonstrates in vitro splicing by asparaginyl endopeptidase of two cofolded polypeptide fragments (A- and B-chains) corresponding to Concanavalin A precursors. This ligation of A- and B-chains was enzyme-, temperature- and pH-dependent. The 9-residue extension at the C-terminus of A-chain is essential for splicing. To test whether correct cofolding and an intervening spacer are required for maturation, recombinant A- and B-chains without spacer sequence were purified and refolded separately. Asparaginyl endopeptidase was again absolutely required for ligation of non-cofolded A- and B-chains. Correct folding is crucial to form an active structure, but is less important for enzyme-mediated splicing. Protein splicing of two-chain forms of precursors was clearly evident from the large decrease in electrophoretic mobility of ligated product. However, interpretation of proteolytic cleavage patterns was difficult. In vivo maturation could be reconstituted in vitro using recombinant pro-Concanavalin A (single-chain, active) with asparaginyl endopeptidase. Enzyme could also splice synthetic peptides corresponding to processing-sequences of precursor proteins. Two short peptides were ligated to form a new longer peptide as indicated by reverse-phase chromatography. It was generally observed that increasing the pH from 5 to 7.5 changed the balance away from proteolytic cleavages (hydrolysis) and towards protein (or peptide) ligation (aminolysis). Promotion of splicing at higher pH indicates that availability of the unprotected (nucleophilic) form of the attacking terminal a-amino group is a major factor in determining product formation. Other factors (substrate conformation, concentrations, temperature and incubation time) may also influence the outcome.

Alzheimer's disease (AD) is a debilitating neurodegenerative disease that affects millions of people worldwide. AD is characterised by the accumulation aggregation and deposition within the brain of amyloid ~ (A~), a 40-42 residue peptide, and hyperphosphorylated Tau. A~ is a cleavage product of the amyloid precursor protein (APP) produced by the sequential cleavage of the ~-secretase, 8ACE1 (~-sjte APP Cleaving enzyme), and the v-secretase. APP can also be proteolytically processed by an alternative pathway where an a-secretase cleaves APP within the A~ region followed by v-cleavage. This precludes the formation of A~. Protein-protein interactions play an important role in regulating protein function; elucidating how the interactions between APP and other proteins affect A~ accumulation in an age-dependent manner is crucial to understanding AD progression. The Apolipoprotein E receptor 2 (ApoEr2) interacts with APP and alters its cleavage, though its exact effect is unclear as it has been shown to both increase and decrease ~-secretase cleavage of APP. To understand the reason for this disparity, 3 different splice variants of ApoEr2 have been expressed in HEK cells and their effects on the levels of APP cleavage products assessed. The isoforms chosen were a full length form (ApoEr2 FL), a form lacking the exon 5 and 15 encoded regions (ApoEr2 1:::.5,15), and a form lacking exon 18 (ApoEr2 1118) which encodes a cytoplasmic proline rich domain. These were chosen as all 3 isoforrns are brain expressed and have been used by different groups in investigating the effects of ApoEr2 on APP processing. It was found that 2 isoforms of ApoEr2 (ApoEr2 FL and 1:::.5,15) caused a reduction in ~- and a-cleavage of APP of -50%, where as ApoEr2 l::.18 increased ~-cleavage -200% whilst having no effect on a- cleavage, To identify the mechanism behind the variation between the isoforms effect on APP Cleavage, the post translational processing of ApoEr2 has been investigated, ApoEr2 was identified as being both N- and O-glycosylated and differences between the isoforms of ApoEr2 in the extent of fully glycosylatyed protein were identified. ApoEr2 l::.5,15 was produced as a fully glycosylated protein. With ApoEr2 FL and 1118 a partially glycosylated protein is also detectable at -25% of total ApoEr2 1:.18 and 40% of total ApoEr2 FL ApoEr2 is also shed from the plasma membrane and there were differences in the shedding of the isoforms of ApoEr2, with ApoEr2 1:.5,15 shed to a lower level than ApoEr2 FL or 1:::.18. The enzyme responsible for the shedding of ApoEr2 has been identified as ADAM10, an enzyme also involved in cleaving APP. The subcellular localisation of ApoEr2 was also investigated and increased detection of ApoEr2 in the early endosome may explain the isoform specific effect of ApoEr2 on APP cleavage. This work has shown for the first time that different splice forms of ApoEr2 have radically different effects on APP proteolytic processing, The fact that splice forms of a protein can have such different effects on APP processing shows that the regulation of APP cleavage is exceedingly complex. This work has also significantly extended what is known about the post translational processing of ApoEr2 with the identification of the enzyme responsible for the shedding of ApoEr2 and has shown that there is substantial variation in post translational processing of the different isoforms of ApoEr2.

Abstract Rheumatoid arthritis (RA) is an autoimmune disease causing inflammation of synovial joints, which may lead to permanent changes in cartilage and bone tissues. RA patients have antibodies binding to citrullinated and also to carbamylated proteins. Antibodies binding to citrulline are associated with more severe disease progression and may already appear years before clinical disease onset. Citrulline and the lysine carbamylation product, homocitrulline, are similar in structure. Citrullinated proteins have been studied in RA and in neurological diseases, but researchers have been unaware of the effect of homocitrulline in citrulline detection methods. The purpose of the present study was to clarify the features of protein-bound citrulline and homocitrulline in relation to research done on citrullination and in immunological reactions related to rheumatoid arthritis. In the first study of this thesis the confounding role of homocitrulline in citrulline detection was shown. In the first and second study the features of experimentally induced antibodies were assessed. The antibodies induced with citrulline- and homocitrulline-containing protein structures were shown to react both with the ureido groups and the protein structures. The antibodies were able to distinguish between citrulline and homocitrulline in the same sequence even though binding to both. In the third study the simultaneous presence of citrulline and homocitrulline in RA synovial tissue was shown. In conclusion, considering the simultaneous presence of citrulline and homocitrulline and the binding features of the experimentally induced antibodies, homocitrulline could have a yet unsolved role in RA. Secondly, the existence of homocitrulline should be borne in mind in studies on citrullination
Tiivistelmä Nivelreuma on niveltulehduksen aiheuttava autoimmuunitauti, joka voi johtaa pysyviin muutoksiin nivelen rusto- ja luukudoksessa. Nivelreumaa sairastavilla esiintyy vasta-aineita sitrullinoituneita ja karbamyloituneita proteiineja vastaan. Sitrulliiniin sitoutuvia vasta-aineita voi esiintyä elimistössä jo vuosia ennen taudin puhkeamista, ja niiden esiintyminen on yhdistetty vaikeampaan taudinkuvaan. Sitrulliini ja lysiinin karbamylaatiotuote homositrulliini ovat rakenteellisesti samankaltaisia. Proteiinien sitrullinaatiota on tutkittu nivelreumassa ja neurologisissa taudeissa, mutta homositrulliinin olemassaoloa tai sen vaikutusta tutkimusmenetelmiin ei ole huomioitu. Tämän tutkimuksen tarkoituksena oli selvittää proteiineihin sitoutuneiden sitrulliinin ja homositrulliinin ominaisuuksia aikaisempiin tutkimuksiin ja nivelreuman immunologisiin reaktioihin liittyen. Tässä tutkimuksessa homositrulliinin osoitettiin häiritsevän sitrulliinin tunnistamista. Ensimmäisessä ja toisessa osatyössä aiheutettiin kokeellisesti vasta-aineita sitrulliinia ja homositrulliinia sisältävillä proteiinirakenteilla. Vasta-aineiden havaittiin reagoivan sekä ureidoryhmän että proteiinirakenteen kanssa. Vasta-aineet pystyivät erottamaan sitrulliinin ja homositrulliinin toisistaan samassa rakenteessa, vaikka sitoutuivat kumpaankin. Kolmannessa osatyössä osoitettiin, että sitrulliinia ja homositrulliinia esiintyy samanaikaisesti nivelreumapotilaan tulehtuneessa nivelkalvossa. Tutkimus osoitti, että sitrulliinin ja homositrulliinin samanaikainen esiintyminen ja kokeellisesti aiheutettujen vasta-aineiden ominaisuudet huomioiden homositrulliinilla voi olla jokin toistaiseksi selvittämätön rooli nivelreumassa. Homositrulliinin olemassaolo on syytä huomioida sitrullinaatiota tutkittaessa

La méthylation de la lysine 9 de l’histone 3 (H3K9), établie par les lysine méthyltransférases (KMTs) SETDB1, SUV39H1, G9A et GLP, représente un mécanisme épigénétique central dans la régulation du destin cellulaire. En particulier, la methylation d’H3K9 est directement impliquée dans la formation de l’hétérochromatine et l’extinction des gènes. Notre laboratoire a montré que les principales KMTs (SETDB1, G9A, GLP et SUV39H1) spécifiques de H3K9 forment un méga-complexe impliqué dans la répression transcriptionnelle, probablement via une coopération pour établir les différents niveaux de méthylation. Néanmoins, la régulation des complexes de H3K9 KMT n’est jusqu’à présent pas bien comprise. Il est à noter que des modifications post-traductionnelles (PTM) ont été impliquées dans la régulation des fonctions des KMTs. Dans ce contexte, mon projet visait à comprendre comment la méthylation de SETDB1 régulerait son activité (incorporation dans des complexes, interaction avec ses partenaires, recrutement à la chromatine). Le but étant d’établir quel impact auraient ces modifications de SETDB1 sur la formation de l’hétérochromatine, l’expression des gènes et la régulation du destin cellulaire. SETDB1 est cruciale lors du développement et de la différençiation cellulaire. De plus, SETDB1 est essentielle pour la pluripotence et le renouvellement des cellules souches embryonnaires murines (mESC). L’inactivation génique de ou KO de Setdb1 est létal au stade préimplantatoire à 7,5 jours post-coïtum (dpc). En plus des histones, SETDB1 méthyle d’autres protéines comme UBF, ING2 et p53. Mes résultats montrent notamment, que SETDB1 s’autométhyle sur les lysines K1170 et K1178 localisées dans le domaine catalytique SET. SETDB1 et SUV39H1 coordonnent l’établissement et la maintenance de H3K9me3 dans l’hétérochromatine péricentromérique constitutive et co-régulent de nombreuses cibles génomiques dans l’hétérochromatine, dont les éléments transposables comme les Long Interspersed Nuclear Elements (LINEs) et les rétrovirus endogènes (ERVs). Comme SUV39H1 est une triméthyltransférase qui utilise H3K9me1 ou H3K9me2 comme substrat primaire, SETDB1 pourrait probablement fournir les mono- ou di-méthyl H3K9. Mes résultats suggèrent un modèle dans lequel l’auto-méthylation de SETDB1 est pré-requise à la trans-méthylation subséquente par SUV39H1. Ce mécanisme pourrait réguler non seulement l’interaction physique entre SETDB1 et SUV39H1, via le chromodomaine de SUV39H1, mais aussi leur coopération dans l’établissement et la maintenance des blocs (grands domaines) d’hétérochromatine et l’extinction des éléments transposables, au moins dans les cellules souches. Ainsi, nous souhaitons mieux comprendre comment le « dialogue » entre ces deux H3K9 KMT majeures, SETDB1 et SUV39H1, est impliqué dans leurs interactions et leurs recrutements aux loci cibles
Histone H3 lysine 9 (H3K9) methylation, which is established by the lysine methyltransferases (KMTs) SETDB1, SUV39H1, G9A and GLP, is a central epigenetic mechanism involved in cell fate regulation. In particular, H3K9 methylation is directly involved in heterochromatin formation and gene silencing. Our lab showed that the main H3K9 KMTs (SETDB1, G9A, GLP and SUV39H1) form a functional megacomplex involved in transcriptional silencing, probably via the cooperative establishment of the different H3K9 methylation levels. However, up to now, the regulation of the H3K9 KMT complexes is not fully understood. Interestingly, post-translational modifications (PTMs) have been implicated in the regulation of H3K9 KMT functions. In this, my PhD thesis aimed to decipher how methylation of SETDB1, regulates its activity (complex formation, interaction with partners, recruitment to chromatin), which ultimately could impact on heterochromatin formation, gene expression and cell fate regulation. SETDB1 is crucial during development and cellular differentiation. Moreover, SETDB1 is essential in mouse embryonic stem cells (mESCs) pluripotency and self-renewal, Setbd1 KO is lethal at the peri-implantation stage at 7.5 days postcoitum (dpc). Beside histones, SETDB1 is also able to methylate other proteins (e.g. UBF, ING2, p53). Notably, my current data show that SETDB1 undergoes (auto)methylation on the lysines K1170 and K1178 located inside its catalytic SET domain. SETDB1 and SUV39H1 coordinate the establishment and maintenance of H3K9me3 at constitutive pericentromeric heterochromatin and co-regulate many genomic targets within heterochromatin, including transposable elements, such as long interspersed nuclear elements (LINEs) and endogenous retroviruses (ERVs). Since SUV39H1 is a H3K9 tri-methyltransferase that uses H3K9me1 or H3K9me2 as a primary substrate, SETDB1 could probably provide mono- or di-methylated H3K9. Interestingly, my results point to a model in which SETDB1 auto-methylation paves the path to a subsequent trans-methylation by SUV39H1. This mechanism could regulate not only the SETDB1/SUV39H1 physical interaction (via the SUV39H1 chromodomain), but also cooperation in the establishment and maintenance of both heterochromatin blocks (large domains) and transposable elements (TEs) silencing, at least in ES cells. Thus, we would like to better understand how the crosstalk between these two key H3K9 KMTs, SETDB1 and SUV39H1, occurs in terms of interaction and recruitment to target loci

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by a loss of voluntary movement over time, leading to paralysis and death. While 10% of ALS cases are inherited or familial (FALS), the majority of cases (90%) are sporadic (SALS) with unknown etiology. Approximately 20% of FALS cases are genetically linked to a mutation in the anti-oxidizing enzyme, superoxide dismutase (SOD1). SALS and FALS are clinically indistinguishable, suggesting a common pathogenic mechanism exists for both types. Since such a large number of genetic mutations in SOD1 result in FALS (>170), it is reasonable to suspect that non-genetic modifications to SOD1 induce structural perturbations that result in ALS pathology as well. In fact, misfolded SOD1 lacking any genetic mutation was identified in end stage spinal cord tissues of SALS patients using misfolded SOD1-specific antibodies. In addition, this misfolded WT SOD1 found in SALS tissue inhibits axonal transport in vitro, supporting the notion that misfolded WT SOD1 exhibits toxic properties like that of FALS-linked SOD1. Indeed, aberrant post-translational modifications, such as oxidation, cause WT SOD1 to mimic the toxic properties of FALS-linked mutant SOD1. Based on these data, I hypothesize that modified, misfolded forms of WT SOD1 contribute to SALS disease progression in a manner similar to FALS linked mutant SOD1 in FALS. The work presented in this dissertation supports this hypothesis. Specifically, one common misfolded form of SOD1 is defined and exposure of this toxic region is shown to enhance SOD1 toxicity. Preventing exposure, or perhaps stabilization, of this “toxic” region is a potential therapeutic target for a subset of both familial and sporadic ALS patients. Further, the possibility of exploiting this misfolded SOD1 species as a biomarker is explored. For example, an over-oxidized SOD1 species was identified in peripheral blood mononuclear cells (PBMCs) from SALS patients that is reduced in controls. Moreover, 2-dimensional gel electrophoresis revealed a more negatively charged species of SOD1 in PBMCs of healthy controls greatly reduced in SALS patients. This species is hypothesized to be involved in the degradation of SOD1, further implicating both misfolded SOD1 and altered protein homeostasis in ALS pathogenesis.

Caenorhabditis elegans possui quatro genes de kpcs (kex2/subtilisin-like proprotein convertases): kpc-1, kpc-2/egl-3, kpc-3/aex-5, kpc-4/bli-4. Em C. elegans, dois dos quatro polipeptídeos de vitelogenina encontrados dentro dos ovócitos, YP115 e YP88, se originam a partir de um precursor polipeptídico (VIT-6) clivado pós-traducionalmente após o motivo RGKR. Nematóides transgênicos foram produzidos com construções repórteres transcricionais de GFP. Foi verificada expressão de kpc-1 tanto em neurônios quanto em células musculares e intestinais. Esses dados, aliados aos dados da literatura para os outros genes kpc de C. elegans, sugerem o envolvimento de KPC-1 no processamento de VIT-6, que é secretada por células intestinais. Ensaios de Western-blot compararam o processamento de VIT-6 em nematóides selvagens, mutantes e knock-down por RNAi para os diferentes genes kpc. A análise de nematóides mutantes e knock-down por RNAi combinado para os outros três genes de convertase de C. elegans confirmou a redundância da atividade dessas enzimas no processamento de VIT-6.
Four kpc genes are found in the Caenorhabditis elegans genome: (kex2/subtilisin-like proprotein convertases): kpc-1, kpc-2/egl-3, kpc-3/aex-5, kpc-4/bli-4. Two of the four vitellogenin polypeptides, YP115 and YP88, originate from a precursor, VIT-6. VIT-6 is cleaved post-translationally after the RGKR motif. Transgenic worms carrying GFP transcription reporter constructs were produced. Expression of kpc-1 has been localized to neurons as well as muscular and intestinal cells. These data, together with the ones available from the literature for the other kpc genes, suggest the involvement of KPC-1 in the processing of VIT-6, which is secreted from intestinal cells. Western-blot analysis compared the pattern of VIT-6 processing in wild-type, mutants and RNAi-treated worms for the other kpcs. Analysis of worms treated by combined RNAi confirmed the redundancy of KPCs in VIT-6 processing.

O estudo de venenos de artrópodes é de grande interesse para melhorar os tratamentos contra envenenamentos e oferece uma ótima ferramenta para melhor compreensão dos sistemas nervoso e imunológico, coagulação sanguínea e respostas inflamatórias. As abelhas são um dos animais venenosos mais estudados e a elucidação do seu proteoma é de interesse na elucidação de reações tóxicas e alérgicas a ferroadas. O número de acidentes envolvendo estes insetos é crescente, tendo ultrapassado 20.000 notificações entre 2001 e 2006 em todo o país e, apesar disso, não há um tratamento específico para estas vítimas, nem mesmo uma identificação completa dos antígenos presentes nesse veneno. O perfil protéico descrito até então apresenta cerca de 40 proteínas. O objetivo deste trabalho foi identificar o perfil protéico do veneno de abelhas utilizando a união da abordagem proteômica e da cromatografia de afinidade. Identificar também as proteínas alergênicas deste veneno e algumas modificações pós-traducionais como fosforilação e glicosilação. Além disso, um soro antiveneno específico foi produzido e sua ação neutralizadora testada. O veneno de abelhas foi separado por cromatografia de afinidade utilizando o soro antiveneno imobilizado em coluna de Sepharose 4B. Para identificação das proteínas foram utilizadas técnicas de 2D-SDS-PAGE, MALDI TOF/TOF e nanoESI-LC/MS-MS. Ensaios de Western Blotting foram realizados para identificar as proteínas alergênicas e fosforiladas. A utilização da cromatografia de afinidade permitiu a identificação 2 de proteínas pouco abundantes. Foram identificadas 54 proteínas, dentre as quais 9 nunca haviam sido descritas neste veneno, como MRJP-2, alfaglicosidase, transferinas, proteases, quinases e um inibidor de protease. Após a identificação destas proteínas foi possível propor um provável mecanismo de ação deste veneno. Dentre as proteínas identificadas como alergênicas, a MRJP-8 foi identificada pela primeira vez, juntamente com fatores relacionados ao PDGF e VEGF. Os resultados dos ensaios de neutralização de atividades citotóxicas, hemolíticas e miotóxicas mostraram a eficiência do soro antiveneno produzido. Chegou-se a um volume de 5,7 mL de soro antiveneno necessários para neutralizar a ação tóxica provocada por 100 ferroadas de abelhas. Este valor está na mesma faixa de eficiência dos melhores antivenenos (ofídicos, aracnídicos e escorpionídicos) produzidos no Brasil e no mundo. O lote de soro antiveneno produzido mostrou resultados satisfatórios para ser utilizado nos testes clínicos
The aim of this work was to identify the protein profile of honeybee venom, and detect allergenic proteins and post-translational modifications. Furthermore specific antivenom was produced and potency tests were performed in order to check its power of neutralization of toxic activities of venom. They were identified 54 proteins, 9 that have never been reported before in this venom. After identification of these proteins it was possible to outline a feasible mechanism of action of venom. For the first time MRJP-8, transferrin, PDGF and VEGF factors were identified as allergenic. Results of neutralization of citotoxic, hemolytic and myotoxic activities showed the efficacy of antivenom that had satisfactory results to be tested in clinical assay

The beta-amyloid peptide characteristic of the lesions of Alzheimer's disease (AD) is derived from the amyloid precursor protein (APP), a single transmembrane-spanning protein with several alternatively-spliced variants, some of which contain a Kunitz protease inhibitory (KPI) domain. Although intracellular localisation of APP has been described in many cell types, it has not been characterised in NTera 2 (NT2) neurones, which are the best available model of human CNS neurones. Here, the subcellular distributions of APP and APLP2 (amyloid precursor-like protein 2) were demonstrated, by indirect immunocytochemistry, to be overlapping but not the same in both NT2 stem cells and neurones. No obvious differences were apparent when comparing the locations of either APP or APLP2 in stem cells versus neurones, APLP2 being restricted to the region of the Golgi apparatus, and APP extending into compartments approaching the cell membrane, including growth cones of neurones. Therefore, no clear differences in intracellular routing of these proteins were identified, immunocytochemically, in human CNS-type neurones compared with stem cells, which represent non-neuronal cells. This work emphasised the need to use antibodies that distinguish between APP and APLP2 (which does not contain the beta-amyloid sequence) in studies of APP processing and amyloidogenesis, since only APP was concentrated in compartments beyond the Golgi apparatus. Heat-shock and no feeding had no immunocytochemically detectable effects on APP or APLP2 distributions or 13-amyloid production in NT2 cells. Although a preliminary investigation to establish a protocol by which NT2 cells can be studied by electron microscopy produced only scant cellular material, more recent publications have shown that slight variations on methods tested here give a successful protocol. The expression of KPI-containing APP in human brains has only been described previously in terms of its mRNA. The ratio of KPI-/non-KPI-APP mRNA appears to be elevated in AD (Tanaka et al., 1989; Johnston et al., 1996). A novel polyclonal antibody (Ab993), specific for the KPI-domain epitope, was characterised for use in immunohistochemistry using paraffin-embedded human brain sections. Immunohistochemical staining was enhanced significantly by reduction of sulphydryl bonds with 2-mercaptoethanol, followed by alkylation of the reduced bonds with sodium iodoacetate. Microwaving of sections also enhanced immunolabelling, by a mechanism that was additive to reduction and alkylation. Incubation with 80% formic acid did not increase immunolabelling. KPI-containing protein distribution in normal and AD human brains was characterised by indirect immunohistochemistry. KPI-APP was concentrated mainly in pyramidal cells of the temporal and visual neocortex. In Alzheimer's disease there was a significantly increased incidence of cellular staining for KPI-APP. KPI-containing protein was closely related to the pathology of AD. It was found in association with the tangle-bearing population of neurones, blood vessels including those affected by cerebro-vascular amyloid, within the neuropil and in association with plaques. This evidence corroborates that supplied previously by mRNA data and studies of hAPP transgenic mice in highlighting the importance of this isoform of APP in the pathogenesis of AD.The cytokine transforming growth factor-beta1 (TGF-beta1) is upregulated in AD brains, but its mRNA expression has not been characterised. TGF-beta1 and hAPP(V717F) bigenicmice show accelerated development of AD-like pathology compared with hAPP(V717F) singly transgenic mice (Wyss-Coray et al, 1997). It has been suggested that elevated TGF-beta1 could increase expression of KPI-containing APP isoforms in AD and/or upregulate CNS extracellular matrix proteins that may promote amyloid deposition. TGF-beta1 mRNA in AD and control frozen human brain sections was quantified by in situ hybridization histochemistry. A modest, significant increase in TGF-beta1 mRNA was found in AD temporal cortex and white matter compared to controls, supporting previous immunohistochemical studies.

Abstract Cellular signaling by G protein-coupled receptors (GPCRs) governs a wide array of physiological functions throughout the body. The human δ-opioid receptor (hδOR) is a GPCR that modulates the sensation of pain and mood and has great potential for the treatment of pain and a variety of neurological disorders. A common single-nucleotide polymorphism (SNP) in the extracellular N-terminal tail of hδOR changes Phe to Cys at position 27. Using various biochemical and cell biological methods, the study demonstrates that several events during receptor biosynthesis and cell surface delivery are affected by the SNP. These events participate in the multifaceted regulation of the receptor and modulate receptor behavior at the cell surface. Two distinct pathways were shown to scrutinize the quality of the synthesized hδOR in the endoplasmic reticulum (ER) and target some for degradation in N-glycan-dependent and -independent ways. The hδORCys27 that matures inefficiently required N-glycan-mediated interactions with the lectin-chaperone calnexin to be expressed in a fully functional form at the cell surface, whereas the N-glycan-independent pathway was sufficient for hδORPhe27. For both variants, the N-glycan-independent quality control, which is likely to operate as a back-up pathway, led to a more rapid export from the ER and receptors at the cell surface that were less stable. Receptor dimerization emerged as an important regulatory step for receptor cell surface delivery. In co-transfected cells, interactions between the newly-synthesized variants led to the retention and subsequent ER-associated degradation of hδORPhe27. This dominant-negative attenuation of hδORPhe27 cell surface expression by hδORCys27 may have unpredictable consequences for opioid signaling in heterozygous individuals. Finally, the study shows that N-acetylgalactosamine (GalNAc)-type O-glycosylation catalyzed in the Golgi modulates hδOR expression at the cell surface by enhancing receptor stability and inhibiting constitutive downregulation. The modification of Ser residues in the receptor N-terminus by GalNAc-transferase 2 was affected by the SNP, which presents another distinction in the cellular processing of the two variants. The findings highlight the importance of the biosynthetic pathway in the regulation of GPCR behavior and pave way for strategies for treatments targeting GPCRs at this level
Tiivistelmä Solujenvälisellä viestinnällä on keskeinen tehtävä kehon kaikissa toiminnoissa. δ-opioidireseptori (δOR) on solusignalointiin erikoistuneen kalvoproteiiniperheen (G-proteiiniin kytketyt reseptorit) jäsen, joka ohjaa kivuntuntemusta ja mielialoja. Sitä pidetään mahdollisena lääkekehityksen kohteena paitsi kivunlievityksen, myös useiden neurologisten häiriöiden hoidossa. δOR ilmenee kahtena polymorfisena muotona sen solunulkoisessa osassa tapahtuneen aminohappomuutoksen vuoksi (Phe27Cys). Työssä tutkittiin reseptorin glykosylaatiota ja dimerisaatiota, jotka säätelevät sen prosessointia, käyttäytymistä ja toimintaa. Käyttäen useita biokemiallisia ja solubiologisia menetelmiä työssä osoitettiin polymorfian vaikuttavan useisiin prosessointivaiheisiin ja muokkaavan siten reseptorin viestintää. Proteiinien laadunvalvontakoneiston havaittiin säätelevän reseptorin siirtymistä endoplasmakalvostolta solun pinnalle kahdella eri mekanismilla ohjaten osan reseptoreista hajotukseen. Toisin kuin Phe27-variantin, tehottomasti kypsyvän Cys27-variantin laadunvalvonta on riippuvainen reseptoriin liittyvistä N-glykaaneista ja näihin sitoutuvasta kaitsijaproteiinista, kalneksiinista. Reseptorivariantit, joista N-glykaanit puuttuvat, siirtyvät nopeammin solukalvolle, mutta ne ovat epästabiileja ja häviävät nopeasti solun pinnalta. Vaihtoehtoinen N-glykaaneista riippumaton laadunvalvontamekanismi sallii myös inaktiivisen Cys27-variantin pääsyn solun pinnalle. Varianttien dimerisoitumisen osoitettiin säätelevän niiden kuljetusta soluissa. Cys27-variantin havaittiin sitoutuvan Phe27-varianttiin aikaisessa biosynteesivaiheessa ja ohjaavan osan siitä hajotukseen. Tällä voi olla suuri merkitys opioidiviestinnässä molempia alleeleja kantavilla henkilöillä. Työssä havaittiin myös GalNAc-transferaasi-2-entsyymin ohjaavan Golgin laitteessa tapahtuvaa reseptorin O-glykosylaatiota. Se glykosyloi reseptorin solunulkoisen osan seriinitähteitä (Ser6, Ser25, Ser29), stabiloiden siten solun pinnan reseptoreita ja tehostaen niiden viestintää. Lisäksi havaittiin eroja varianttien O-glykosylaatiossa, mikä voi osaltaan selittää varianttien ilmentymisessä todettuja eroja. Tutkimus luo uutta tietoa biosynteesireitin merkityksestä G-proteiiniin kytkettyjen reseptorien säätelyssä sekä antaa pohjaa keinoille, joilla tätä voitaisiin hyödyntää farmakologisesti

Ma Kit Wan.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2005.
Includes bibliographical references (leaves 189-206).
Abstracts in English and Chinese.
Acknowledgements --- p.i
Table of Contents --- p.iii
Abstract --- p.xi
摘要 --- p.xiv
Abbreviation List --- p.xv
List of Figures --- p.xvii
List of Tables --- p.xxiii
Chapter Chapter 1 --- Introduction --- p.1
Chapter 1.1 --- Ubiquitination --- p.1
Chapter 1.1.1 --- Ubiquitin --- p.1
Chapter 1.1.2 --- Ubiquitin Pathway --- p.3
Chapter 1.1.3 --- Functions of Ubiquitination --- p.5
Chapter 1.1.4 --- Ubiquitin Like Proteins --- p.8
Chapter 1.2 --- SUMO Proteins --- p.10
Chapter 1.2.1 --- SUMO Isoforms --- p.10
Chapter 1.2.2 --- SUMO Structure --- p.11
Chapter 1.3 --- Sumoylation --- p.14
Chapter 1.3.1 --- Functions of Sumoylation --- p.14
Chapter 1.3.1.1 --- General Functions of Sumoylation --- p.15
Chapter 1.3.1.2 --- Function of Sumoylation on Transcription Factors
Chapter 1.3.1.3 --- Specific Function of SUMO-2/3 Conjugation
Chapter 1.3.2 --- Sumoylation Pathway --- p.19
Chapter 1.4 --- E3 Ligases in Sumoylation --- p.24
Chapter 1.4.1 --- Types and Functions of E3 Ligases --- p.23
Chapter 1.4.2 --- Structure of PI AS --- p.23
Chapter 1.4.3 --- Function of PI AS --- p.27
Chapter 1.5 --- Aims of Study --- p.29
Chapter Chapter 2 --- Materials & Methods --- p.30
Chapter 2.1 --- Polymerase Chain Reaction (PCR) Screening of Multiple Human Tissue cDNA (MTC´ёØ) Panel --- p.30
Chapter 2.1.1 --- Primer Design --- p.30
Chapter 2.1.2 --- Semi-quantitative PCR --- p.31
Chapter 2.1.2.1 --- Human MTC´ёØ Panel --- p.31
Chapter 2.1.2.2 --- PCR --- p.32
Chapter 2.2 --- DNA Cloning --- p.34
Chapter 2.2.1 --- "Amplification of El, E3 (PIAS), PIAS1 Fragments" --- p.34
Chapter 2.2.1.1 --- Primer Design --- p.34
Chapter 2.2.1.2 --- PCR --- p.36
Chapter 2.2.1.3 --- Purification of PCR Product --- p.37
Chapter 2.2.2 --- Restriction Digestion --- p.37
Chapter 2.2.3 --- Ligation --- p.40
Chapter 2.2.4 --- Transformation --- p.40
Chapter 2.2.4.1 --- Preparation of Chemically Competent Cells'(DH5α) --- p.40
Chapter 2.2.4.2 --- Transformation of Ligation Product --- p.41
Chapter 2.2.5 --- Plasmid Preparation --- p.42
Chapter 2.2.6 --- Screening for Recombinant Clones --- p.43
Chapter 2.2.7 --- Sequencing of Recombinant Plasmid --- p.43
Chapter 2.3 --- Subcellular Localization Study --- p.45
Chapter 2.3.1 --- Midi Scale Plasmid Preparation --- p.45
Chapter 2.3.2 --- Transfection of GFP Recombinant Plasmids --- p.46
Chapter 2.3.2.1 --- Cell Culture of WRL-68 & HepG2 Cell Lines --- p.46
Chapter 2.3.2.2 --- LipofectAMINE Based Transfection --- p.47
Chapter 2.3.3 --- Immunostaining of Endogenous SUMO-1 & -2/-3 --- p.48
Chapter 2.3.4 --- Nucleus Staining by DAPI --- p.48
Chapter 2.3.5 --- Fluorescent Microscopic Visualization --- p.49
Chapter 2.3.6 --- Western Blotting --- p.49
Chapter 2.3.6.1 --- LipofectAMINE Based Transfection --- p.49
Chapter 2.3.6.2 --- Protein Extraction --- p.50
Chapter 2.3.6.3 --- Protein Quantification --- p.51
Chapter 2.3.6.4 --- SDS-PAGE Analysis --- p.51
Chapter 2.3.6.5 --- GFP Fusion Proteins Detection --- p.52
Chapter 2.4 --- Two-Dimensional Gel Electrophoretic Analyses --- p.54
Chapter 2.4.1 --- Sample Preparation --- p.54
Chapter 2.4.1.1 --- Protein Extraction from the Nucleus --- p.54
Chapter 2.4.1.2 --- Clean Up of Extracted Nuclear Fraction --- p.55
Chapter 2.4.2 --- First Dimensional Isoelectric Focusing (IEF) --- p.55
Chapter 2.4.3 --- Second Dimension SDS-PAGE --- p.57
Chapter 2.4.3.1 --- SDS-PAGE Analysis --- p.57
Chapter 2.4.3.2 --- Silver Staining --- p.58
Chapter 2.4.4 --- Image Analysis --- p.59
Chapter 2.4.5 --- Protein Identification by Mass Spectrometry --- p.60
Chapter 2.4.5.1 --- Sample Preparation --- p.60
Chapter 2.4.5.2 --- Data Acquisition --- p.62
Chapter 2.4.5.3 --- Data Analysis of Protein Fingerprinting --- p.62
Chapter 2.5 --- Confirmation of the Differentially Expressed Proteins by RT-PCR & Western Blotting --- p.63
Chapter 2.5.1 --- RT-PCR Analysis --- p.63
Chapter 2.5.1.1 --- RNA Extraction --- p.63
Chapter 2.5.1.2 --- First Strand cDNA Synthesis --- p.64
Chapter 2.5.1.3 --- Normalization of cDNA Template --- p.64
Chapter 2.5.1.4 --- PCR Amplification of the Target Genes --- p.65
Chapter 2.5.2 --- Western Blotting --- p.66
Chapter 2.6 --- Expression of Human PIAS and PIAS1 Fragments in Prokaryotic System --- p.67
Chapter 2.6.1 --- Preparation of Competent Cells --- p.67
Chapter 2.6.2 --- Small Scale Expression --- p.67
Chapter 2.6.2.1 --- Transformation --- p.67
Chapter 2.6.2.2 --- IPTG Induced Protein Expression --- p.68
Chapter 2.6.3 --- Large Scale Expression of PIAS1 Fragments --- p.70
Chapter 2.6.3.1 --- Transformation --- p.70
Chapter 2.6.3.2 --- IPTG Induced Protein Expression --- p.70
Chapter 2.6.4 --- Purification Trial of MBP-PIAS1-321-410 --- p.71
Chapter 2.6.4.1 --- Binding of Amylose Resin & On Column Cleavage (with Low Concentration of DTT) --- p.71
Chapter 2.6.4.2 --- Elution from the Amylose Resin & Cleavage (with Low Concentration of DTT) --- p.73
Chapter 2.6.4.3 --- Elution from the Amylose Resin & Cleavage (with High Concentration of DTT) --- p.73
Chapter 2.6.4.4 --- Purification of PIAS1-321-410 by Size ExclusionChromatography --- p.73
Chapter 2.6.5 --- Purification of MBP-PIAS1 Fragments --- p.74
Chapter 2.6.5.1 --- Purification by Affinity Column (Amylose) --- p.74
Chapter 2.6.5.2 --- Amylose Resin Regeneration --- p.74
Chapter 2.6.5.3 --- Purification by Both Affinity and Ion Exchange (Heparin) --- p.75
Chapter 2.6.5.4 --- Regeneration of Heparin Column --- p.76
Chapter 2.6.5.5 --- Purification by Size Exclusion Chromatography --- p.76
Chapter 2.6.5.6 --- Regeneration of Size Exclusion Chromatography --- p.77
Chapter 2.6.6 --- Co-expression & Purification of PIAS1 Fragment with E2 (Ubc9) --- p.77
Chapter 2.6.6.1 --- Co-transformation of pMAL-PIASl (Fragments) & pET-Ubc9 --- p.77
Chapter 2.6.6.2 --- Co-expression of PIAS1 Fragments & Ubc9 --- p.78
Chapter 2.6.6.3 --- Purification by Affinity Column (Amylose Resin) --- p.78
Chapter 2.6.6.4 --- Purification by Both Affinity & Ion Exchange (Heparin) --- p.79
Chapter 2.6.6.5 --- Purification by Size Exclusion Chromatography --- p.79
Chapter 2.6.7 --- Urea Treatment for the Purification of PIAS 1 Fragments --- p.80
Chapter 2.6.7.1 --- Transformation --- p.80
Chapter 2.6.7.2 --- IPTG Induced Protein Expression --- p.80
Chapter 2.6.7.3 --- Purification by Affinity Column (Amylose Resin) --- p.80
Chapter 2.6.7.4 --- Purification by Both Affinity & Ion Exchange (Heparin) --- p.80
Chapter 2.6.7.5 --- Purification by Size Exclusion Chromatography --- p.81
Chapter Chapter 3 --- Results --- p.82
Chapter 3.1 --- Tissue Distribution of Human PIAS Genes --- p.82
Chapter 3.1.1 --- Determination of the Number of Cycles for PCR --- p.82
Chapter 3.1.2 --- General Expression Pattern of All PIAS Genes --- p.82
Chapter 3.1.3 --- Tissue Distribution of PIAS1 --- p.83
Chapter 3.1.4 --- Tissue Distribution of PIAS3 --- p.83
Chapter 3.1.5 --- Tissue Distribution of PIASxa --- p.83
Chapter 3.1.6 --- Tissue Distribution of PIASxp --- p.84
Chapter 3.1.7 --- Tissue Distribution of PIASy --- p.84
Chapter 3.2 --- Subcellular Localization of SUMO Pathway Components --- p.90
Chapter 3.2.1 --- Overexpression Confirmation --- p.90
Chapter 3.2.2 --- Multiple Bands Detected After Overexpression of EGFP- SUMO-1 --- p.91
Chapter 3.2.3 --- Subcellular Localization of EGFP --- p.94
Chapter 3.2.4 --- Subcellular Localization of El Subunits --- p.94
Chapter 3.2.5 --- Subcellular Localization of E2 (Ubc9) --- p.95
Chapter 3.2.6 --- Subcellular Localization of PIAS Proteins --- p.95
Chapter 3.2.7 --- Subcellular Localization of PIAS1 Fragments --- p.96
Chapter 3.2.8 --- Subcellular Localization of SUMO-1 --- p.97
Chapter 3.3 --- Differential Protein Expression Pattern after Transient Transfection of SUMO-1 --- p.112
Chapter 3.3.1 --- Protein Expression Profiles after Transient Transfection
Chapter 3.3.2 --- Identification of the Differential Expressed Proteins --- p.113
Chapter 3.4 --- Confirmation of Differentially Expressed Proteins in Cells Overexpressing SUMO-1 --- p.124
Chapter 3.4.1 --- RT-PCR Analyses --- p.124
Chapter 3.4.1.1 --- Downregulation of RNA Transcript of hnRNP A2/B1 isoform B1 --- p.124
Chapter 3.4.1.2 --- No Significant Change in the Transcription Level of UDG --- p.125
Chapter 3.4.2 --- Western Blotting --- p.128
Chapter 3.4.2.1 --- Upregulation of hnRNP A2/B1 at the Protein Level --- p.128
Chapter 3.4.2.2 --- Different Molecular Weight of hnRNP A2/B1 Was Detected --- p.129
Chapter 3.4.2.3 --- Upregulation of UDG at the Protein Level --- p.129
Chapter 3.5 --- Expression & Purification of Human PIAS Proteins & PIAS1 Fragments --- p.133
Chapter 3.5.1 --- Expression of Human PIAS Proteins --- p.133
Chapter 3.5.2 --- Expression of PIAS1 Fragments --- p.135
Chapter 3.5.3 --- A Trial of Purification of MBP-PIAS1-321-410 --- p.137
Chapter 3.5.3.1 --- On Column Cleavage of MBP Tag --- p.137
Chapter 3.5.3.2 --- Cleavage after Elution --- p.137
Chapter 3.5.3.3 --- High Concentration of DTT Used --- p.138
Chapter 3.5.3.4 --- Separation of the Cleaved and Non Cleaved Proteins --- p.138
Chapter 3.5.4 --- Purification of the PIAS 1 Fragments --- p.141
Chapter 3.5.4.1 --- Purified by Affinity Column (Amylose Resin) --- p.141
Chapter 3.5.4.2 --- Purified by Heparin Column --- p.141
Chapter 3.5.4.3 --- Purified by Gel Filtration --- p.143
Chapter 3.5.5 --- Co-expression & Purification of PIAS1 Fragments & E2 --- p.147
Chapter 3.5.5.1 --- Co-expression of PIAS1 Fragments & E2 --- p.147
Chapter 3.5.5.2 --- Co-purification of PIAS1 Fragments & E2 Amylose --- p.147
Chapter 3.5.5.3 --- Co-purification of PIAS1 Fragments & E2 by Heparin --- p.148
Chapter 3.5.5.4 --- Co-purification of PIAS 1 Fragments with Ubc9 by Gel Filtration --- p.148
Chapter 3.5.6 --- Urea Treatment for Purification of PIAS1 Fragments --- p.153
Chapter 3.5.6.1 --- Purification by Amylose Resin --- p.153
Chapter 3.5.6.2 --- Purification by Heparin --- p.153
Chapter 3.5.6.3 --- Purification by Gel Filtration --- p.154
Chapter Chapter 4 --- Discussion --- p.157
Chapter 4.1 --- Tissue Specificity of PIAS Proteins --- p.157
Chapter 4.1.1 --- Principle of Tissue Specificity Study --- p.157
Chapter 4.1.2 --- Importance of Sumoylation --- p.158
Chapter 4.1.3 --- Role of Sumoylation in Reproduction --- p.159
Chapter 4.1.4 --- Functional Role of Sumoylation in Other Tissue --- p.160
Chapter 4.2 --- Subcellular Localization of SUMO Pathway --- p.162
Chapter 4.2.1 --- SUMO Conjugation Occurs in the Nucleus --- p.162
Chapter 4.2.2 --- Does Sumoylation Occur Outside the Nucleus --- p.163
Chapter 4.2.3 --- Dots-like Structure Formed by the PIAS --- p.164
Chapter 4.2.4 --- SAP Domain and PINIT Motif Are Not Essential for Nuclear Targeting --- p.165
Chapter 4.2.5 --- Signal Involves in the Formation of Nuclear Speckles --- p.167
Chapter 4.3 --- Differentially Expressed Proteins under SUMO-1 Overexpression --- p.169
Chapter 4.3.1 --- Increase in High Molecular Weight Proteins --- p.169
Chapter 4.3.2 --- Upregulation of hnRNP A2/B1 & UDG in Protein Level --- p.170
Chapter 4.3.3 --- Variants of hnRNP A2/B1 Formed --- p.172
Chapter 4.3.4 --- Possibility of Sumoylation on hnRNP A2/B1 isoform B1 & UDG --- p.172
Chapter 4.3.5 --- Possible Roles of SUMO-1 on hnRNP A2/B1 isoform B1 --- p.174
Chapter 4.3.6 --- Mechanism of Sumoylation on mRNA Processing --- p.175
Chapter 4.3.7 --- Possible Roles of SUMO-1 on UDG --- p.176
Chapter 4.3.8 --- Important of SUMO on Genome Integrity --- p.178
Chapter 4.3.9 --- Sumoylation and Carcinogenesis --- p.178
Chapter 4.4 --- Protein Purification of the Human PIAS Proteins & PIAS1 Fragments --- p.180
Chapter 4.4.1 --- Low Expression Level & Solubility of the PIAS Proteins --- p.180
Chapter 4.4.2 --- High Expression Level & Solubility of PIAS 1 Fragments --- p.181
Chapter 4.4.3 --- Incorrect Disulfide Bond Formation of the PIAS1 Fragments --- p.182
Chapter 4.4.4 --- MBP-PIAS1 Fragments Formed Soluble Aggregates --- p.182
Chapter 4.4.5 --- A Low Concentration of Urea Cannot Dissociate the Soluble Aggregates --- p.183
Chapter 4.4.6 --- Aggregation May Weaken the Interaction between the PIAS1 Fragments & Ubc9 --- p.184
Chapter 4.5 --- Conclusion --- p.185
Chapter 4.6 --- Future Perspectives --- p.187
Chapter 4.6.1 --- Identification of the Role of SUMO Interacting Motif in the Nuclear Speckle Formation --- p.187
Chapter 4.6.2 --- Investigation of Sumoylation on Liver Cancer --- p.187
Chapter 4.6.3 --- Optimization of the Expression & Purification of the PIAS Proteins --- p.188
References --- p.189
Appendix --- p.207

Dissertação de Mestrado em Bioquímica apresentada à Faculdade de Ciências e Tecnologia
The Leber’s Hereditary Optic Neuropathy (LHON) is a rare mitochondrial disorder characterized by the loss of retinal ganglion cells (RGCs), which are responsible for the conduction of the visual information to the cerebral cortex. This loss leads to the degeneration of the optic nerve resulting in a sudden loss of vision. The major confirmed genetic causes of this disorder are mitochondrial DNA (mtDNA) mutations in genes encoding subunits belonging to the complex I of the mitochondrial respiratory chain (MRC), namely MT-ND1 (m.3460G>A), MT-ND4 (m.11778G>A) and MT-ND6 (m.14484T>C). The presence of these mutations do not totally explain the disease phenotype, since the existence of individuals carrying homoplasmic mtDNA mutations without disease manifestation have been reported. There are several factors including nuclear genetic modifiers that have been suggested to be influencers of the phenotype manifestation. The presence of nuclear gene variants in subunits and proteins involved in the mitochondrial protein import and processing of imported precursor proteins may contribute as genetic modifiers in LHON. To assess this possibility, a search for the mentioned genetic variants in whole-exome sequencing data was performed and the prediction of functional impact of the relevant variants was evaluated using bioinformatics’ tools. This analysis resulted in the identification of the promising c.280C>T and c.170delA/c.172_176delGGCAC variants from MIPEP and TOMM20L gene, respectively, in a LHON individual with the m.14484T>C mutation. These variants were confirmed using two additional methods, namely Sanger sequencing and PCR-RFLP.The implications at the protein level have been investigated in a preliminary study. Limitations at the level of tissue specificity of Tom20L protein expression did not allow the evaluation of the impact of the genetic variants identified in the protein expression. Further experimental validation of the genetic variants is required for the clarification of its pathogenicity, but the results suggest that the alteration c.280C>T of MIPEP gene may have an additional effect on the pathogenicity of the variant m.14484T>C.
A Neuropatia Ótica Hereditária de Leber (LHON) é uma doença mitocondrial rara caracterizada pela perda de células ganglionares da retina, responsáveis pelo envio da informação visual para o córtex cerebral. A perda destas células resulta na degenerescência do nervo ótico e, consequentemente, na perda súbita da visão. As causas genéticas que comprovam a presença desta doença são mutações em genes mitocondriais que codificam subunidades do complexo I da cadeia respiratória mitocondrial, nomeadamente MT-ND1 (m.3460G>A), MT-ND4 (m.11778G>A) e MT-ND6 (m.14484T>C). A presença destas mutações não explica totalmente o fenótipo da doença, visto que a existência de indivíduos portadores de mutações do mtDNA homoplásmicas, sem manifestação da doença tem sido reportada. Existem vários fatores incluindo fatores genéticos nucleares, que têm sido sugeridos como moduladores da manifestação da doença. Um destes fatores genéticos poderá ser a presença de variantes em genes nucleares, que codificam subunidades e proteínas envolvidas na importação de proteínas para a mitocôndria e no processamento pós-tradução e importação. No sentido de avaliar esta possibilidade, foi realizada a pesquisa destas variantes em dados de sequenciação de exoma total e foi avaliada a previsão do impacto funcional das variantes relevantes. A análise resultou na identificação de 3 variantes promissoras: c.170delA e c.172_176delGGCAC no gene TOMM20L e c.280C>T no gene MIPEP, num doente afetado com LHON portador da mutação patogénica m.14484T>C. As variantes identificadas foram confirmadas por dois métodos adicionais, sequenciação de Sanger e PCR-RFLP. As implicações das variantes genéticas ao nível da proteína foram investigadas num estudo preliminar. Limitações ao nível da especificidade da expressão tecidular da proteína Tom20L não permitiram avaliar o impacto das variantes genéticas identificadas ao nível da expressão da proteína. A validação funcional e experimental adicional das variantes genéticas identificadas será necessária para clarificar a sua patogenicidade, mas os resultados sugerem que a alteração c.280C>T do gene MIPEP poderá ter um efeito potenciador da patogenicidade da variante m.14484T>C.
Outro - Projecto financiado por Fundos FEDER através do Programa Operacional Factores de Competitividade - COMPETE 2020 e por fundos nacionais através da FCT - Fundação para a Ciência e a Tecnologia no âmbito do projecto Estratégico com referência atribuída pelo COMPETE: POCI-01-0145-FEDER-007440

In this study, the brain of senescence accelerated mouse prone 8 (SAMP8) mice model was adopted to investigate PTMs state (especially methylation patterns) of core histones (H2A, H2B, H3 and H4). Seven methylated sites (H3K24, H3K27, H3K36, H3K79, H3R128, H4K20 and H2A R89) were detected by tandem matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) analysis. The methylation of H3K27 and H3K36 demonstrated a modulating relationship and methylated H3K27 might contribute to the hypermethylation state and gene repression in aged brain. Western blotting results showed that mono-methylated H4K20 decreased during SAMP8 mice aging and di-methylated H3K79 decreased in the brain of 12-month-old SAMP8 mice compared with age-matched senescence accelerated-resistant mouse (SAMR1) control. Di-methylated H3K79 could express in neuron cells of cerebral cortex and hippocampus. Whereas, the number of H3K79 methylation negative cells was higher in the cortex of 12-month old SAMP8 mice than that of age-matched control SAMR1 mice. Chromatin immunoprecipitation (ChIP) result indicated homeodomain transcription factor Pbx1 isoform 1 (Pbx1), transcription factors and transcriptional regulator proteins, such as T-box isoform 20, TetR family precursor BAZ2B and ribosomal protein, were recruited to methylated H3K79 site. Therefore, a model of methylated H3K79 on gene transcriptional regulation was proposed. Furthermore, the consequences of decreased H3K79 methylation in Neuro-2a (N2a) cells were investigated via transfection with Dot1 (disruptor of telomeric silencing) siRNA. After transfection, N2a cells displayed shorter neurite and less dendrite. Proteomic change in the N2a cells provided convincing evidence for the multi-function of decreased H3K79 methylation on transcriptional regulation, protein translation and folding, stress response and DNA breaks repair, which would contribute to brain dysfunction during neurodegenerative disease or aging.
Nowadays, many countries including China are experiencing aging populations. Aging has become the major risk factor for many diseases, such as neurodegenerative disease. The studies on the role of epigenetics in the aging process have grown tremendously in recent years. However, no systematic investigations have provided the information on histone post-translational modifications (PTMs) in aged brain and the roles of histone PTMs in brain aging are still unknown.
This study gave a new insight into the link between histone PTMs and brain aging. It could provide the experimental evidence for future studies and help us to better understand aging or neurodegenerative disease at epigenetic level. Furthermore, it could benefit for setting up the strategies for epigenetic therapy to neurodegenerative disease.
Wang, Chunmei.
Adviser: Ngai Saiming.
Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: .
Thesis (Ph.D.)--Chinese University of Hong Kong, 2009.
Includes bibliographical references (leaf 136).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstract also in Chinese.

Jiang, Lili.
Thesis (Ph.D.)--Chinese University of Hong Kong, 2011.
Includes bibliographical references (leaves 215-235).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstract also in Chinese.

Le régulateur transcriptionnel BAP1 est une déubiquitinase nucléaire (DUB) dont le substrat est l’histone H2A modifiée par monoubiquitination au niveau des residus lysines 118 et 119 (K118/K119). Depuis les dernières années, BAP1 emerge comme un gene suppresseur de tumeur majeur. En effet, BAP1 est inactivé dans un plethore de maladies humaines héréditaires et sporadiques. Cependant, malgré l’accumulation significative des connaissances concernant l’occurrence, la pénétrance et l’impact des défauts de BAP1 sur le développement de cancers, ses mécanismes d’action et de régulation restent très peu compris. Cette étude est dédiée à la caractérisation moléculaire et fonctionnelle du complexe multi-protéique de BAP1 et se présente parmi les premiers travaux décrivant sa régulation par des modifications post-traductionnelles. D’abord, nous avons défini la composition du corps du complexe BAP1 ainsi que ses principaux partenaires d’interaction. Ensuite, nous nous sommes spécifiquement intéressés a investiguer d’avantage deux principaux aspects de la régulation de BAP1. Nous avons d’abord décrit l’inter-régulation entre deux composantes majeures du complexe BAP1, soit HCF-1 et OGT. D’une manière très intéressante, nous avons trouvé que le cofacteur HCF-1 est un important régulateur des niveaux protéiques d’OGT. En retour, OGT est requise pour la maturation protéolytique de HCF-1 en promouvant sa protéolyse par O-GlcNAcylation, un processus de régulation très important pour le bon fonctionnement de HCF-1. D’autre part, nous avons découvert un mécanisme unique de régulation de BAP1 médiée par l’ubiquitine ligase atypique UBE2O. en effet, UBE2O se caractérise par le fait qu’il s’agit aussi bien d’une ubiquitine conjuratrice et d’une ubiquitine ligase. UBE2O, multi-monoubiquitine BAP1 au niveau de son domaine NLS et promeut son exclusion du noyau, le séquestrant ainsi dans le cytoplasme. De façon importante, nos travaux ont permis de mettre de l’emphase sur le rôle de l’activité auto-catalytique de chacune de ces enzymes, soit l’activité d’auto-déubiquitination de BAP1 qui est requise pour la maintenance de sa localisation nucléaire ainsi que l’activité d’auto-ubiquitination d’UBE2O impliquée dans son transport nucléo-cytoplasmique. De manière significative, nous avons trouvé que des défauts au niveau de l’auto-déubiquitination de BAP1 due à des mutations associées à certains cancers indiquent l’importance d’une propre regulation de cette déubiquitinase pour les processus associés à la suppression de tumeurs.
BAP1 is a nuclear deubiquitinating enzyme (DUB) that acts as a transcription regulator and a DUB of nucleosomal histone H2AK119. In the recent years, it has become clear that BAP1 is a major tumor suppressor, inactivated in a plethora of hereditary and sporadic human malignancies. Although, we now accumulated a significant body of knowledge in respect to the occurrence, penetrance and impact of BAP1 disruption in cancer, its mechanism of action and regulation remained poorly defined. This work is dedicated to the biochemical and functional characterization of the BAP1 multiprotein complex and presents one of the first cases regarding its regulation by post-translational modifications. First, we defined the initial composition of the BAP1 complex and its main interacting components. Second, we specifically focused on two aspects of BAP1 regulation. We described the cross regulation between the two major components of the complex namely HCF-1 and OGT. We found that HCF-1 is important for the maintenance of the cellular levels of OGT. OGT, in turn, is required for the proper maturation of HCF-1 by promoting O-GlcNAcylation-mediated limited proteolysis of its precursor. Third, we discovered an intricate regulatory mechanism of BAP1 mediated by the atypical ubiquitin ligase UBE2O. UBE2O multi-monoubiquitinates BAP1 on its NLS and promotes its exclusion from the nucleus. Importantly, our work emphasises the role of the autocatalytic activity of both enzymes namely the auto-deubiquitination activity of BAP1, required for the maintenance of nuclear BAP1 and the auto-ubiquitination of UBE2O implicated in its nucleocytoplasmic transport. Significantly, we found that auto-deubiquitination of BAP1 is disrupted by cancer-associated mutations, indicating the involvement of this process in tumor suppression.

N-glycosylation is the most common eukaryotic post-translational modification, impacting on protein stability, folding, and protein-protein interactions. More broadly, N-glycans play biological roles in reaction kinetics modulation, intracellular protein trafficking, and cell-cell communications. The machinery responsible for the initial stages of N-glycan assembly and processing is found on the membrane of the endoplasmic reticulum. Following N-glycan transfer to a nascent glycoprotein, the enzyme Processing α-Glucosidase I (GluI) catalyzes the selective removal of the terminal glucose residue. GluI is a highly substrate-specific enzyme, requiring a minimum glucotriose for catalysis; this glycan is uniquely found in biology in this pathway. The structural basis of the high substrate selectivity and the details of the mechanism of hydrolysis of this reaction have not been characterized. Understanding the structural foundation of this unique relationship forms the major aim of this work. To approach this goal, the S. cerevisiae homolog soluble protein, Cwht1p, was investigated. Cwht1p was expressed and purified in the methyltrophic yeast P. pastoris, improving protein yield to be sufficient for crystallization screens. From Cwht1p crystals, the structure was solved using mercury SAD phasing at a resolution of 2 Å, and two catalytic residues were proposed based upon structural similarity with characterized enzymes. Subsequently, computational methods using a glucotriose ligand were applied to predict the mode of substrate binding. From these results, a proposed model of substrate binding has been formulated, which may be conserved in eukaryotic GluI homologs.