Scienze biologiche

Settori scientifico disciplinari compresi nell'area 5:



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  • Publication
    Structural and Biochemical study of human RECQ4
    (Università degli studi di Trieste, 2015-04-28)
    Mojumdar, Aditya
    Onesti, Silvia
    RecQ helicases belong to a ubiquitous family of DNA unwinding enzymes that are essential to maintain genome stability by acting at the interface between DNA replication, recombination and repair. Humans have five different paralogues of RecQ helicases namely RecQ1, BLM, WRN, RecQ4 and RecQ5. This work focuses on the structural and biochemical study of human RecQ4. Germ-line mutations in the RECQ4 gene give rise to three distinct human genetic disorders (Rothmund-Thomson, RAPADILINO and Baller-Gerold syndromes). Despite the important roles of RecQ4 in various cellular processes, RecQ4 have never been fully characterized. In addition to the helicase domain, RecQ4 has a unique N-terminal part that is essential for viability and is constituted by a region homologous to the yeast Sld2 replication initiation factor, followed by a cysteine-rich region, predicted to fold as a Zn knuckle. A part of this work focuses on the structural and biochemical analysis of both the human and Xenopus RecQ4 cysteine-rich regions, and shows by NMR spectroscopy that the Xenopus fragment does indeed assumes the canonical Zn knuckle fold, whereas the human sequence remains unstructured, consistent with the mutation of one of the Zn ligands. Both the human and Xenopus Zn knuckles bind to a variety of nucleic acid substrates, with a preference for RNA. We also investigated the effect of an additional Sld2 homologous region upstream the Zn knuckle. In both the human and Xenopus system, the presence of this region strongly enhances binding to nucleic acids. These results reveal novel possible roles of RecQ4 in DNA replication and genome stability. Recently the catalytic core of RecQ4 has been predicted to include RecQ-like-C-terminal (RQC) domain at the C-terminus of the helicase domain, similar to other RecQ helicases. This domain is composed of a Zn-binding region and a winged helix (WH) domain. Another part of this thesis centers on the structural and biochemical characterization of the catalytic core of RecQ4 including the helicase and RQC domain. The results provide an insight in the Zn binding ligands present in the RQC domain that plays a role in DNA binding and unwinding activity of the protein. Also the presence of the characteristic aromatic residue at the tip of the WH β hairpin and its role in DNA binding and unwinding has been established. Finally, it provides a low resolution SAXS model of the catalytic core of RecQ4.
      1059  1136
  • Publication
    (Università degli studi di Trieste, 2015-04-28)
    Di Girolamo, Filippo Giorgio
    Biolo, Gianni
    Dietary proteins are the source of the amino acids required by the body for tissue growth and maintenance. The Population Reference Intake (PRI) for proteins, as defined by the European Food Safety Authority (EFSA) for healthy adults, including the elderly, is 0.83 g/kg body weight/day. This amount is defined on the net balance of body protein (or “nitrogen balance”, given by the difference between dietary nitrogen intake and losses) equivalent to 0.66 g/kg/day plus a safety factor for interpersonal variability and differences in proteins quality of mixed diets. The PRI, however, is the minimum daily amount of protein needed to maintain the nitrogen balance and avoid a progressive loss of lean body mass in healthy people with moderate physical activity. Therefore nitrogen balance may not be adequate to define protein requirement in adults and especially in ageing characterized by loss of muscle mass and function (sarcopenia). Furthermore until recently the prevalent idea was that a protein intake above PRI had no further benefits and on the contrary could impair health. These believes are now under discussion, diets with higher protein intake have been shown beneficial in the prevention and treatment of conditions such as sarcopenia, COPD and type 2 diabetes mellitus. There is a need of more precise methods to define protein requirement. AIM. The aim of the present thesis is to investigate in human healthy volunteers new biomarkers adequate to define optimal protein intake. Recent studies have determined protein needs by measuring whole-body protein metabolism using stable labeled isotope-amino acids. METHODS. Our research group has applied two different metabolic methods based on the most widely used tracer, i.e. D5-Phe stable isotope, in two experimental bed rest campaigns (FP7 PLANHAB and INTERREG PANGaA) in healthy volunteers. BR is a suitable model to investigate physiologic adaptation to inactivity. MAIN RESUTLTS. FP7 PLANHAB. We applied the stable isotope infusion technique, to assess the effect of physical inactivity and/or hypoxic condition on whole body protein turnover as previously described in Biolo et al 2008. Chronic hypoxia has been associated with an overall reduction in protein synthesis and in total plasma and skeletal muscle protein content. During the PLANHAB study we investigated, through a crossover randomization, the net effects of 10 days normobaric hypoxia (4000 mt.), associated with either ambulatory conditions or BR, in 11 young (age 24±4 yr), healthy and normal weight male subjects maintained on eucaloric diets. Main results. Hypoxia in ambulatory conditions significantly decreased whole body protein turnover by reducing both protein synthesis (-8±2%) and protein degradation (-8±3%). Hypoxia during bed rest did not caused significant changes in protein metabolism. INTERREG PANGaA. The skeletal muscle loss in aging is caused mainly by the “anabolic resistance” i.e. the inadequate increase in the rate of protein synthesis in response to nutritional-metabolic stimuli, including exercise, protein and amino acid intake as well as insulin and insulin-like growth factor stimulation. As a consequence, the net protein balance becomes negative leading to sarcopenia. The effects of ageing on the anabolic resistance induced by inactivity are poorly investigated. During the PANGeA study we had the opportunity to perform the second documented experimental BR in in healthy elderly volunteers and the first comparing aged with young subjects. To evaluate the anabolic resistance associated with ageing and inactivity, we enrolled 7 young (23±1yr) and 8 elderly (59±1yr) normal weight individuals, in a 14-d experimental BR protocol. We replaced our previous infusion method with a new, simpler, safer and quicker technique, by which tracers are given orally instead of parenterally, the all procedure is completed in two hours, instead of 6, and only two blood draws versus 7 are sufficient. Main results. At baseline parameters of anabolic sensitivity were comparable between young and elderly individuals. The anabolic resistance significantly increased after BR in both groups (bed-rest effect p<0.01), with a statistically significant bed-rest×group interaction (p=0.01). Anabolic resistance increased significantly in elderly (18.5%±7.3%) more than in young (5.2%±9.4%) subjects. DISCUSSION. In the PLANHAB study, hypoxia in ambulatory conditions reduced by the same level both protein synthesis and catabolism, as measured by isotope infusions, suggesting an adaptive mechanism: the lower energy production and availability induced by hypoxia associated with ambulatory condition. These modifications could not have been revealed by the use of nitrogen balance method, showing the relevance of more sophisticated analysis. The direct evaluation of the muscle protein metabolism through an infusion of stable-labeled isotope tracer, considered the golden standard methodology, gave us, in the PLANHAB study, reliable results in the early protein metabolism changes during hypoxia and/or BR. This method however has the limit of being complex, onerous and invasive, therefore being unsuitable for clinical evaluation. In the PANGeA study we could confirm the presence of a reduced sensitivity to anabolic stimuli in the elderly population compared to the young men. The elderly subjects are therefore, more at risk to develop changes of protein metabolism induced by inactivity. The simpler, timesaving and less invasive method we have developed for the PANGeA study, on the other hand, could be applied to a wider ranges of experimental conditions and clinical settings.
      908  948
  • Publication
    Expression and biochemical studies of the human MCM2-7 helicase
    (Università degli studi di Trieste, 2015-04-28)
    Di Crescenzio, Patrizia
    Onesti, Silvia
    The MCM helicase plays a key role in DNA replication, as it unwinds the double helix, providing a single stranded template for the DNA polymerases. MCM genes are found both in Eukaryotes and Archaea. Eukaryotic genomes present at least 6 different MCM genes, which share significant sequence similarity. The six different polypeptides encoded by MCM genes assemble into a heteroexamer, characterized by a toroidal conformation. Each protein comprises 3 domains: the N-terminal domain binds DNA and is essential for hexamerization; the C-terminal domain folds as a winged-helix motif and may have a regulatory role; the central AAA+ domain is the catalytic core of the helicase that couples the ATP hydrolysis with DNA unwinding. Since the eukaryotic MCM complex is difficult to produce in large amounts, most of the available structural information derive from studies on the archaeal model. For eukaryotic MCM proteins no crystal structure has been determined yet, while low resolution reconstructions by electron microscopy are available only for S. cerevisiae and D. melanogaster complexes. The MCM proteins are present only in proliferating cells and are highly expressed in malignant cancer cells and pre-cancerous cells undergoing malignant transformation. Their expression has been compared with routinely used proliferation markers, showing that they are suitable candidates as biomarkers for cancer in clinical practice. Therefore, a detailed knowledge of their structure and function is a crucial pre-requisite for their potential role in cancer diagnosis and therapy. Based on bioinformatics analysis and structural information from archaeal MCM proteins, fragments corresponding to the AAA+ domains and N-terminal domains of the human MCM helicase were designed and cloned separately into different expression vectors. Various expression strategies were explored and constructs were tested for protein expression under a variety of conditions. AAA+ domains showed a severe problem of solubility: most of them precipitated during purification, aggregated or, in the best cases, precipitated after tag removal. The attempt to co express or co purify the subunits together didn’t improve the solubility of the proteins, also in presence of ATP analogues. The N-terminal domains were soluble and expressed at high levels. The co purification of the six fragments led to the assembly of two different oligomeric states compatible with a double and a single hexamer. Both oligomers fit the biological role of the protein, expected to be loaded onto the double stranded DNA as double hexamer and to unwind as single hexamer. DNA binding experiments were carried out by fluorescence polarization: both assemblies are able to bind different DNA substrates (single strand, double strand, fork) with micromolar dissociation constants, consistent with the DNA binding affinities reported for the archaeal MCM complex. A preliminary biophysical characterization performed with different techniques (thermal shift analysis, circular dichroism, multi angle light scattering, small angle X-ray scattering) revealed that the putative single hexamer is properly folded but quite unstable, as it showed the tendency to disassemble into smaller oligomers. In contrast, the putative double hexamer showed a higher stability, but still a degree of inhomogeneity which didn’t allow a structural study of its architecture. Nevertheless, the high yield of our purification protocol represents a promising starting point for further optimization, allowing crystallography and other structural studies.
      800  782
  • Publication
    (Università degli studi di Trieste, 2015-04-28)
    Ruggeri, Naomi
    Del Sal, Giannino
    The Hippo signalling pathway is tumour suppressor cascade with a central role in the regulation of fundamental cellular biological processes, such as cell proliferation, apoptosis, organ size control and stem cell functions. The Hippo pathway transduces external signals that come to the cell into the nucleus, where it can control the expression of specific target genes, mainly involved in cell proliferation and differentiation. The Hippo pathway is an inhibitory pathway that control by phosphorylation and inhibition Yes-associated protein (YAP) coactivator, one of the two nuclear effectors of this signalling, involved in the regulation of proliferation and organ size. As consequence, deregulation of Hippo tumor suppressor pathway or hyperactivation of its downstream effectors is often associated with formation, development and tumour dissemination. Consistently, YAP is often over-expressed in a broad range of different tumours and it has aberrant activity in breast cancer as well as in several other human carcinomas. Up-regulation of YAP activity increases stem cell self-renewal in normal and cancer stem cells. In this work we describe the identification of a new hormonal-dependent layer for YAP regulation in breast cancer by the glucocorticoids and we analyze the mechanisms through which this regulation occurs. We found that Glucocorticoid Receptor (GR) binds directly the YAP promoter and induces the transcription of YAP mRNA after GC stimulation in cancer cells. Moreover, GC lead to efficient YAP de-phosphorylation and transcriptional activation, in a transcription-independent manner, by inducing actin cytoskeleton reorganization. Importantly, inhibition of the GR by means of RU486 (GR competitive antagonist) strongly blunted the expansion of the cancer stem cell pool in breast cancer cells by blunting the GR/YAP axis.
      1054  1528
  • Publication
    Telomere regulation by microRNAs in human breast cancer
    (Università degli studi di Trieste, 2015-04-28)
    Dinami, Roberto
    Schoeftner, Stefan
    Nei vertebrati i telomeri sono strutture specializzate localizzate all’estremità dei cromosomi costituito da sequenze ripetute TTAGGG. L’ accorciamento progressivo del telomero ad ogni divisione cellulare porta ad una disfunzione telomerica e all’attivazione della risposta di danno al DNA alle estremità cromosomiche. L’attivazione del segnale di danno al DNA provoca senescenza o apoptosi, fenomeni legati all’invecchiamento. Per mantenere la funzione del telomero il complesso della telomerasi, costituito dalla componente ad RNA (hTERC) e dalla subunità catalitica (hTERT), aggiunge ripetizioni telomeriche in cellule con alto potenziale replicativo. Due complessi principali sono coinvolti nella regolazione dei telomeri: il complesso shelterin e il complesso della telomerasi. Shelterin è costituito da sei principali proteine: TRF1, TRF2, POT1, TPP1, TIN2 e RAP1, e controlla vari aspetti della funzione telomerica come la lunghezza telomerica, la ricombinazione e la protezione da fattori di risposta al danno al DNA. l’evasione dalla senescenza replicativa raggiunta con la riattivazione della telomerasi, è un passaggio chiave nel processo di tumorigenesi, come osservato nel 90% dei tumori umani. Inoltre, aumentano anche le dimostrazioni che indicano il ruolo centrale di shelterin nella formazione e nella progressione del cancro. L’espressione del complesso shelterin o della telomerasi è strettamente regolata a livello trascrizionale e post-traduzionale, anche se il ruolo dei miRNAs nella regolazione dei telomeri non è ancora stato studiato. Lo scopo del mio progetto di tesi era quello di identificare miRNAs che controllano l’espressione dei componenti di shelterin o del complesso della telomerasi e valutare la rilevanza clinica di questi miRNAs nel contesto del tumore al seno. Per raggiungere questo obiettivo abbiamo eseguito un high-throughput luciferase reporter screening in cellule HeLa identificando un panello di miRNAs che hanno come bersaglio il 3’UTR di componenti del complesso shelterin (TRF1, TRF2, POT1) o del complesso della telomerasi (TERT, DKC1). Con questo screening abbiamo identificato che l’onco-miRNA miR-155 è un efficiente regolatore di TRF1. Il miR-155 è up-regolato in tutti i tipi di tumore al seno ed alti livelli del miR-155 correlano con una bassa espressione dei livelli proteici di TRF1. Inoltre, abbiamo validato con ulteriori saggi luciferasi il targeting di TRF1 da parte del miR-155 ed abbiamo anche scoperto che il miR-155 controlla l’espressione di TRF1 a livello traduzionale. Soprattutto, la bassa espressione dei livelli di mRNA di TRF1 e la bassa espressione dei geni bersaglio del miR-155 correlano con una ridotta sopravvivenza libera da metastasi distanti (DMFS) e con una ridotta sopravvivenza libera da recidive (RFS) in pazienti con cancro al seno luminale estrogeno-positivo (ER+). Questo indica che il targeting di TRF1 da parte del miR-155 è parte di una signature del miR-155 che determina una scarsa sopravvivenza nel cancro al seno di tipo luminale ER+. Soprattutto abbiamo scoperto che il targeting di TRF1 da parte del miR-155 porta ad un aumento della fragilità telomerica, a fusioni telomeriche dei cromatidi fratelli e all’allungamento dei telomeri. Il nostro lavoro dimostra per la prima volta che la regolazione post-trascrizionale di TRF1, mediata dal miR-155, è un efficiente meccanismo per controllare la fragilità telomerica e l’instabilità genomica nel cancro al seno. Invece, per quanto riguarda i miRNAs che regolano la telomerasi nel cancro al seno, abbiamo studiato il miR-296-5p e il miR-512-5p, i quali bersagliano efficientemente hTERT come mostrato dal high-throughput luciferase reporter screening . Entrambi i miRNAs bersagliano specifiche regioni nel 3’UTR di hTERT portando alla degradazione dell’mRNA di hTERT e alla riduzione dell’attività telomerasica. Dati clinici rivelano che il miR-296-5p e il miR-512-5p sono down-regolati nei tessuti tumorali del seno. Inoltre, abbiamo scoperto che l’aumento dell’espressione di hTERT e l’aumentata espressione dei geni bersaglio del miR-296-5p e del miR-512-5p correlano con una scarsa sopravvivenza in pazienti con cancro al seno di tipo basale. Questo evidenzia l’importanza clinica del miR-296-5p e del miR-512-5p nel cancro al seno (basale). A livello molecolare abbiamo mostrato che il miR-296-5p e il miR-512-5p rallentano la proliferazione cellulare e provocano l’ accorciamento della lunghezza dei telomeri in cellule di tumore al seno basale. Soprattutto, abbiamo scoperto che la ri-espressione epigenetica dei geni del miR-296-5p e del miR-512-5p in cellule di tumore al seno basale, determina l’aumento dell’espressione del miR-296-5p e del miR-512-5p e la conseguente riduzione dell’espressione di hTERT. I nostri dati suggeriscono che l’utilizzo di composti che inducono la ri-espressione del miR-296-5p e del miR-512-5p potrebbe essere una strategia promettente per compromettere i meccanismi di mantenimento dei telomeri e la funzione di hTERT nel cancro al seno. I risultati di questo lavoro dimostrano che i miRNAs rappresentano dei nuovi regolatori della funzione telomerica e hanno un impatto sull’omeostasi dei telomeri nel cancro umano. Questo lavoro identifica i miRNAs come nuovi bersagli per modulare la funzione dei telomeri nelle malattie ad essi correlate come il cancro e l’invecchiamento.
      1259  2181