Ruolo di Clusterina nella progressione del tumore prostatico

Clusterina (CLU) è una proteina ubiquitaria, presente nella maggior parte dei fluidi corporei e implicata in svariati processi fisiologici. Dalla sua scoperta fino ad oggi, CLU è risultata essere una proteina enigmatica, la cui funzione non è ancora stata compresa appieno. Il gene codifica per 3 varianti trascrizionali identificate nel database NCBI con i codici: NM_001831 (CLU 1 in questo lavoro di tesi), NR_038335 (CLU 2 in questo lavoro di tesi) e NR_045494 (CLU 3 in questo lavoro di tesi). Tutte le varianti sono trascritte come pre-mRNA contenenti 9 esoni e 8 introni e si differenziano per l’esone 1, la cui sequenza è unica e caratteristica di ogni variante. Sebbene in NCBI sia annotato che le varianti CLU 2 e CLU 3 non sono codificanti, tramite analisi bioinformatica è stato predetto che da tutti e tre i trascritti possono generarsi proteine di differente lunghezza e localizzazione cellulare. Tra tutte le forme proteiche ipotizzate, l’unica a essere stata isolata e sequenziata è quella tradotta dall’AUG presente sull’esone 2 che dà origine a una proteina di 449 aminoacidi. Il processo di maturazione prevede la formazione di un precursore citoplasmatico (psCLU) che subisce modificazioni post-traduzionali tra cui formazione di ponti disolfuro, glicosilazioni, taglio in due catene denominate β e α prima di essere secreta come eterodimero βα (sCLU) nell’ambiente extracellulare, dove esercita la sua funzione di chaperone ATP-indipendente. Oltre alla forma extracellulare, è possibile osservare una forma intracellulare con localizzazione citosolica la cui funzione non è stata ancora completamente chiarita. Questo lavoro di tesi si è prefissato lo scopo di incrementare le conoscenze in merito ai trascritti CLU 1 e CLU 2 e alla loro regolazione, oltre ad approfondire il ruolo della forma citosolica della proteina in relazione al signaling di NF-kB che svolge un ruolo importante nel processo di sviluppo e metastatizzazione del tumore. Nella prima parte, uno screening di differenti linee cellulari, quali cellule epiteliali di prostata e di mammella, sia normali sia tumorali, fibroblasti di origine polmonare e linfociti di tumore non-Hodgkin, ha permesso di caratterizzare i trascritti CLU 1 e CLU 2. Dall’analisi è emerso che la sequenza di CLU 1 è più corta al 5’ rispetto a quella depositata in NCBI con l’identificativo NM_001831 e il primo AUG disponibile per l’inizio della traduzione è localizzato sull’esone 2. È stato dimostrato che CLU 2, al contrario di quanto riportato in NCBI, è tradotto in proteina a partire dall’AUG presente sull’esone 2, allo stesso modo in cui viene tradotto CLU 1. Inoltre, è stato osservato che i livelli d’espressione dei trascritti variano notevolmente tra le diverse linee cellulari e nelle cellule epiteliali CLU 2 è espressa sempre a bassi livelli. In queste cellule, l’espressione di CLU 2 è silenziata per via epigenetica e la somministrazione di farmaci capaci di rendere la cromatina più accessibile, quali tricostatina A e 5-aza-2’-deossicitidina, è in grado di incrementarne l’espressione. Nella seconda parte, un’analisi bioinformatica seguita da saggi di attività in vitro in cellule epiteliali prostatiche trattate con farmaci epigenetici, hanno permesso di identificare, per la prima volta in uomo, una seconda regione regolatrice denominata P2, capace di controllare l’espressione di CLU 2. Rispetto a P1, il classico promotore di CLU già ampiamente studiato da altri gruppi di ricerca, P2 è un promotore debole, privo di TATA box, che nelle cellule epiteliali prostatiche è silente in condizioni basali e la cui attività incrementa in seguito alla somministrazione di farmaci epigenetici capaci di alterare le modificazioni post-traduzionali delle code istoniche nell’intorno di P2. Ne consegue un rilassamento della cromatina e un successivo aumento di trascrizione di CLU 2. La presenza di un’isola CpG differentemente metilata nell’intorno di P1 spiegherebbe, almeno in parte, i differenti livelli di espressione di CLU che si osservano tra le diverse linee cellulari. Nella terza parte, l’analisi del pathway di NF-kB in un modello sperimentale di tumore prostatico in cui CLU è stata silenziata o sovraespressa, ha permesso di capire come la forma citosolica di CLU abbia un ruolo inibitorio nei confronti dell’attività del fattore trascrizionale NF-kB. CLU inibisce la fosforilazione e l’attivazione di p65, il membro più rappresentativo della famiglia NF-kB, con conseguente riduzione della trascrizione di alcuni geni da esso regolati e coinvolti nel rimodellamento della matrice extracellulare, quali l’urochinasi attivatrice del plasminogeno, la catepsina B e la metallo proteinasi 9. È stato dimostrato che tale inibizione non è dovuta a un’interazione fisica diretta tra CLU e p65, per cui si suppone che CLU interagisca con uno dei componenti più a monte della via di segnalazione responsabile della fosforilazione ed attivazione di p65.

Control of neuronal signalling pathways by CYP46A1, an enzime involved in brain cholesterol homeostasis

Tese de doutoramento, Farmácia (Biologia Celular e Molecular), Universidade de Lisboa, Faculdade de Farmácia, 2016

SnRK1-triggered switch of bZIP63 dimerization mediates the low-energy response in plants

Metabolic adjustment to changing environmental conditions, particularly balancing of growth and defense responses, is crucial for all organisms to survive. The evolutionary conserved AMPK/Snf1/SnRK1 kinases are well-known metabolic master regulators in the low-energy response in animals, yeast and plants. They act at two different levels: by modulating the activity of key metabolic enzymes, and by massive transcriptional reprogramming. While the first part is well established, the latter function is only partially understood in animals and not at all in plants. Here we identified the Arabidopsis transcription factor bZIP63 as key regulator of the starvation response and direct target of the SnRK1 kinase. Phosphorylation of bZIP63 by SnRK1 changed its dimerization preference, thereby affecting target gene expression and ultimately primary metabolism. A bzip63 knock-out mutant exhibited starvation-related phenotypes, which could be functionally complemented by wild type bZIP63, but not by a version harboring point mutations in the identified SnRK1 target sites.

Enzymatic characterization and homology model of a catalytically active recombinant West Nile virus NS3 protease

West Nile Virus (WNV) is a mosquito-borne flavivirus with a rapidly expanding global distribution. Infection causes severe neurological disease and fatalities in both human and animal hosts. The West Nile viral protease (NS2B-NS3) is essential for post-translational processing in host-infected cells of a viral polypeptide precursor into structural and functional viral proteins, and its inhibition could represent a potential treatment for viral infections. This article describes the design, expression, and enzymatic characterization of a catalytically active recombinant WNV protease, consisting of a 40-residue component of cofactor NS2B tethered via a noncleavable nonapeptide (G(4)SG(4)) to the N-terminal 184 residues of NS3. A chromogenic assay using synthetic para-nitroanilide (pNA) hexapeptide substrates was used to identify optimal enzyme-processing conditions (pH 9.5, I < 0.1 M, 30% glycerol, 1 mM CHAPS), preferred substrate cleavage sites, and the first competitive inhibitor (Ac-FASGKR- H, IC50 &SIM; 1 μM). A putative three-dimensional structure of WNV protease, created through homology modeling based on the crystal structures of Dengue-2 and Hepatitis C NS3 viral proteases, provides some valuable insights for structure-based design of potent and selective inhibitors of WNV protease.

Modulation of higher-plant NAD(H)-dependent glutamate dehydrogenase activity in transgenic tobacco via alteration of beta subunit levels

Glutamate dehydrogenase (GDH; EC 1.4.1.2-1.4.1.4) catalyses in vitro the reversible amination of 2-oxoglutarate to glutamate. In vascular plants the in vivo direction(s) of the GDH reaction and hence the physiological role(s) of this enzyme remain obscure. A phylogenetic analysis identified two clearly separated groups of higher-plant GDH genes encoding either the alpha- or beta-subunit of the GDH holoenzyme. To help clarify the physiological role(s) of GDH, tobacco (Nicotiana tabacum L.) was transformed with either an antisense or sense copy of a beta-subunit gene, and transgenic plants recovered with between 0.5- and 34-times normal leaf GDH activity. This large modulation of GDH activity (shown to be via alteration of beta-subunit levels) had little effect on leaf ammonium or the leaf free amino acid pool, except that a large increase in GDH activity was associated with a significant decrease in leaf Asp (similar to 51%, P=0.0045). Similarly, plant growth and development were not affected, suggesting that a large modulation of GDH beta-subunit titre does not affect plant viability under the ideal growing conditions employed. Reduction of GDH activity and protein levels in an antisense line was associated with a large increase in transcripts of a beta-subunit gene, suggesting that the reduction in beta-subunit levels might have been due to translational inhibition. In another experiment designed to detect post-translational up-regulation of GDH activity, GDH over-expressing plants were subjected to prolonged dark-stress. GDH activity increased, but this was found to be due more likely to resistance of the GDH protein to stress-induced proteolysis, rather than to post-translational up-regulation.

Protein adduct species in muscle and liver of rats following acute ethanol administration

Aims: Previous immunohistochemical studies have shown that the post-translational formation of aldehyde-protein adducts may be an important process in the aetiology of alcohol-induced muscle disease. However, other studies have shown that in a variety of tissues, alcohol induces the formation of various other adduct species, including hybrid acetaldehyde-malondialdehyde-protein adducts and adducts with free radicals themselves, e.g. hydroxyethyl radical (HER)-protein adducts. Furthermore, acetaldehyde-protein adducts may be formed in reducing or non-reducing environments resulting in distinct molecular entities, each with unique features of stability and immunogenicity. Some in vitro studies have also suggested that unreduced adducts may be converted to reduced adducts in situ. Our objective was to test the hypothesis that in muscle a variety of different adduct species are formed after acute alcohol exposure and that unreduced adducts predominate. Methods: Rabbit polyclonal antibodies were raised against unreduced and reduced aldehydes and the HER-protein adducts. These were used to assay different adduct species in soleus (type I fibre-predominant) and plantaris (type II fibre-predominant) muscles and liver in four groups of rats administered acutely with either [A] saline (control); [B] cyanamide (an aldehyde dehydrogenase inhibitor); [C] ethanol; [D] cyanamide+ethanol. Results: Amounts of unreduced acetaldehyde and malondialdehyde adducts were increased in both muscles of alcohol-dosed rats. However there was no increase in the amounts of reduced acetaldehyde adducts, as detected by both the rabbit polyclonal antibody and the RT1.1 mouse monoclonal antibody. Furthermore, there was no detectable increase in malondialdehyde-acetaldehyde and HER-protein adducts. Similar results were obtained in the liver. Conclusions: Adducts formed in skeletal muscle and liver of rats exposed acutely to ethanol are mainly unreduced acetaldehyde and malondialdehyde species.

Discovery of an MIT-like atracotoxin family: Spider venom peptides that share sequence homology but not pharmacological properties with AVIT family proteins

This project identified a novel family of six 66-68 residue peptides from the venom of two Australian funnel-web spiders, Hadronyche sp. 20 and H. infensa: Orchid Beach (Hexathelidae: Atracinae), that appear to undergo N- and/or C-terminal post-translational modifications and conform to an ancestral protein fold. These peptides all show significant amino acid sequence homology to atracotoxin-Hvf17 (ACTX-Hvf17), a non-toxic peptide isolated from the venom of H. versuta, and a variety of AVIT family proteins including mamba intestinal toxin 1 (MIT1) and its mammalian and piscine orthologs prokineticin 1 (PK1) and prokineticin 2 PK2). These AVIT family proteins target prokineticin receptors involved in the sensitization of nociceptors and gastrointestinal smooth muscle activation. Given their sequence homology to MITI, we have named these spider venom peptides the MIT-like atracotoxin (ACTX) family. Using isolated rat stomach fundus or guinea-pia ileum organ bath preparations we have shown that the prototypical ACTX-Hvf17, at concentrations up to 1 mu M, did not stimulate smooth muscle contractility, nor did it inhibit contractions induced by human PK1 (hPK1). The peptide also lacked activity on other isolated smooth muscle preparations including rat aorta. Furthermore, a FLIPR Ca2+ flux assay using HEK293 cells expressing prokineticin receptors showed that ACTX-Hvf17 fails to activate or block hPK1 or hPK2 receptors. Therefore, while the MIT-like ACTX family appears to adopt the ancestral disulfide-directed beta-hairpin protein fold of MIT1, a motif believed to be shared by other AVIT family peptides, variations in the amino acid sequence and surface charge result in a loss of activity on prokineticin receptors. (c) 2005 Elsevier Inc. All rights reserved.

Pax9 and Jagged1 act downstream of Gli3 in vertabrate limb development

From early in limb development the transcription factor Gli3 acts to define boundaries of gene expression along the anterior-posterior (AP) axis, establishing asymmetric patterns required to provide positional information. As limb development proceeds, posterior mesenchyme expression of Sonic hedgehog (Shh) regulates Gli3 transcription and post-translational processing to specify digit number and identity. The molecular cascades dependent on Gli3 at later stages of limb development, which link early patterning events with final digit morphogenesis, remain poorly characterised. By analysing the transcriptional consequences of loss of Gli3 in the anterior margin of the E11.5 and E12.5 limb bud in the polydactylous mouse mutant extra-toes (Gli3(Xt/Xt)), we have identified a number of known and novel transcripts dependent on Gli3 in the limb. In particular, we demonstrated that the genes encoding the paired box transcription factor Pax9, the Notch ligand Jagged1 and the cell surface receptor Cdo are dependent on Gli3 for correct expression in the anterior limb mesenchyme. Analysis of expression in compound Shh;Gli3 mutant mouse embryos and in both in vitro and in vivo Shh signaling assays, further defined the importance of Shh regulated processing of Gli3 in controlling gene expression. In particular Pax9 regulation by Shh and Gli3 was shown to be context dependent, with major differences between the limb and somite revealed by Shh bead implantation experiments in the chick. Jagged1 was shown to be induced by Shh in the chick limb and in a C3H10T1/2 cell based signaling assay, with Shh;Gli3 mutant analysis indicating that expression is dependent on Gli3 derepression. Our data have also revealed that perturbation of early patterning events within the Gli3(Xt/Xt), limb culminates in a specific delay of anterior chondrogenesis which is subsequently realised as extra digits. (c) 2005 Elsevier Ireland Ltd. All rights reserved.

The complexity of p53 stabilization and activation

A number of proteins are activated by stress stimuli but none so spectacularly or with the degree of complexity as the tumour suppressor p53 (human p53 gene or protein). Once stabilized, p53 is responsible for the transcriptional activation of a series of proteins involved in cell cycle control, apoptosis and senescence. This protein is present at low levels in resting cells but after exposure to DNA-damaging agents and other stress stimuli it is stabilized and activated by a series of post-translational modifications that free it from MDM2 (mouse double minute 2 but used interchangeably to denote human also), a ubiquination ligase that ubiquitinates it prior to proteasome degradation. The stability of p53 is also influenced by a series of other interacting proteins. In this review, we discuss the post-translational modifications to p53 in response to different stresses and the consequences of these changes.

Evolving noisy oscillatory dynamics in genetic regulatory networks

We introduce a genetic programming (GP) approach for evolving genetic networks that demonstrate desired dynamics when simulated as a discrete stochastic process. Our representation of genetic networks is based on a biochemical reaction model including key elements such as transcription, translation and post-translational modifications. The stochastic, reaction-based GP system is similar but not identical with algorithmic chemistries. We evolved genetic networks with noisy oscillatory dynamics. The results show the practicality of evolving particular dynamics in gene regulatory networks when modelled with intrinsic noise.

Is the generation of neo-antigenic determinants by free radicals central to the development of autoimmune rheumatoid disease?

Biomolecules are susceptible to many different post-translational modifications that have important effects on their function and stability, including glycosylation, glycation, phosphorylation and oxidation chemistries. Specific conversion of aspartic acid to its isoaspartyl derivative or arginine to citrulline leads to autoantibody production in models of rheumatoid disease, and ensuing autoantibodies cross-react with native antigens. Autoimmune conditions associate with increased activation of immune effector cells and production of free radical species via NADPH oxidases and nitric oxide synthases. Generation of neo-antigenic determinants by reactive oxygen and nitrogen species ROS and RNS) may contribute to epitope spreading in autoimmunity. The oxidation of amino acids by peroxynitrite, hypochlorous acid and other reactive oxygen species (ROS) increases the antigenicity of DNA, LDL and IgG, generating ligands for which autoantibodies show higher avidity. This review focuses on the evidence for ROS and RNS in promoting the autoimmune responses observed in diseases rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). It considers the evidence for ROS/RNS-induced antigenicity arising as a consequence of failure to remove or repair ROS/RNS damaged biomolecules and suggests that an associated defect, probably in T cell signal processing or/or antigen presentation, is required for the development of disease.

Contributions to our understanding of T cell physiology through unveiling the T cell proteome

Since the sequencing of the human genome was completed, attention has turned to examining the functionality of the molecular machinery, in particular of protein expression. Differential proteome analysis by two-dimensional electrophoresis has been adopted to study changes in T cell proteomes during T cell activation, and this work is increasing our understanding of the complexity of signals elicited across multiple pathways. The purpose of this review is to summarize the available evidence in the application of proteomic techniques and methodologies to understand T cell receptor activation from lipid raft and cytoskeletal rearrangements, through to signalling cascades, transcription factor modulation and changes in protein expression patterns. These include post-translational modifications, which are not encoded by the genome. © 2007 British Society for Immunology.

Free radicals and redox signalling in T-cells during chronic inflammation and ageing

During chronic inflammation and ageing, the increase in oxidative stress in both intracellular and extracellular compartments is likely to influence local cell functions. Redox changes alter the T-cell proteome in a quantitative and qualitative manner, and post-translational modifications to surface and cytoplasmic proteins by increased reactive species can influence T-cell function. Previously, we have shown that RA (rheumatoid arthritis) T-cells exhibit reduced ROS (reactive oxygen species) production in response to extracellular stimulation compared with age-matched controls, and basal ROS levels [measured as DCF (2',7'-dichlorofluorescein) fluorescence] are lower in RA T-cells. In contrast, exposing T-cells in vitro to different extracellular redox environments modulates intracellular signalling and enhances cytokine secretion. Together, these data suggest that a complex relationship exists between intra- and extra-cellular redox compartments which contribute to the T-cell phenotype.

Production of membrane proteins in yeast

Background Yeast is an important and versatile organism for studying membrane proteins. It is easy to cultivate and can perform higher eukaryote-like post-translational modifications. S. cerevisiae has a fully-sequenced genome and there are several collections of deletion strains available, whilst P. pastoris can produce very high cell densities (230 g/l). Results We have used both S. cerevisiae and P. pastoris to over-produce the following His6 and His10 carboxyl terminal fused membrane proteins. CD81 – 26 kDa tetraspanin protein (TAPA-1) that may play an important role in the regulation of lymphoma cell growth and may also act as the viral receptor for Hepatitis C-Virus. CD82 – 30 kDa tetraspanin protein that associates with CD4 or CD8 cells and delivers co-stimulatory signals for the TCR/CD3 pathway. MC4R – 37 kDa seven transmembrane G-protein coupled receptor, present on neurons in the hypothalamus region of the brain and predicted to have a role in the feast or fast signalling pathway. Adt2p – 34 kDa six transmembrane protein that catalyses the exchange of ADP and ATP across the yeast mitochondrial inner membrane. Conclusion We show that yeasts are flexible production organisms for a range of different membrane proteins. The yields are such that future structure-activity relationship studies can be initiated via reconstitution, crystallization for X-ray diffraction or NMR experiments.

AntigenDB:an immunoinformatics database of pathogen antigens

The continuing threat of infectious disease and future pandemics, coupled to the continuous increase of drug-resistant pathogens, makes the discovery of new and better vaccines imperative. For effective vaccine development, antigen discovery and validation is a prerequisite. The compilation of information concerning pathogens, virulence factors and antigenic epitopes has resulted in many useful databases. However, most such immunological databases focus almost exclusively on antigens where epitopes are known and ignore those for which epitope information was unavailable. We have compiled more than 500 antigens into the AntigenDB database, making use of the literature and other immunological resources. These antigens come from 44 important pathogenic species. In AntigenDB, a database entry contains information regarding the sequence, structure, origin, etc. of an antigen with additional information such as B and T-cell epitopes, MHC binding, function, gene-expression and post translational modifications, where available. AntigenDB also provides links to major internal and external databases. We shall update AntigenDB on a rolling basis, regularly adding antigens from other organisms and extra data analysis tools. AntigenDB is available freely at http://www.imtech.res.in/raghava/antigendb and its mirror site http://www.bic.uams.edu/raghava/antigendb.