1,720,956 research outputs found
Structural studies of protein kinase CK2: Inhibition mechanisms and structure-activity relationships
Full text linkThe subject of this thesis is the protein kinase CK2 which is a family of enzymes that in humans consists of two catalytic subunits, termed CK2α and CK2α’, and one regulatory subunit, CK2β. CK2 is a highly conserved acidophilic Ser/Thr protein kinase, ubiquitously distributed in different cell compartments. CK2 is a member of the superfamily of eukaryotic protein kinases (EPKs), meaning the catalytic subunit is related by sequence homology and structural features to the other 478 kinases of the superfamily.
CK2 shows some singular features like its high constitutive activity and the lack of an defined mode of regulation, which make CK2 unique with respect to the other kinases. With hundreds of substrates, this kinase is involved in several cellular events, resulting essential for the cell viability: CK2β gene knockout in mouse model is lethal even at single cell level, CK2α gene knockout are embryonic lethal at day 10.5 and CK2α' (expressed only in brain and testis) mouse knockout are viable with some defects in spermatogenesis. It participates in cell cycle progression, gene expression, cell growth, and differentiation and embryogenesis. Down-regulation of CK2 leads to apoptosis while abnormal over-activation has been found coupled to several diseases: the clinical relevance of CK2 is that high levels of the protein activity have been detected in a number of cancers, such as head and neck, renal, breast, prostate, lung, and kidney.
A wide spectrum of cell permeable, fairly specific ATP site directed CK2 inhibitors are currently available which are proving useful to dissect its biological functions and which share the property of inducing apoptosis of cancer cells with no comparable effect on their “normal” counterparts. One of these, CX-4945, has recently entered clinical trials for the treatment of advanced solid tumors, Castelman’s disease and multiple myeloma.
CK2 is considered constitutively active enzyme and, unlike many other protein kinases, it does not require phosphorylation for activation. The mechanism of regulation of CK2 is not firmly established yet, however it is clear that it differs from those commonly utilized by other protein kinases.
Dozens of crystal structures of CK2 have been solved and highlighted the structural features of the main CK2 entities, the catalytic subunit CK2α, the regulatory subunit CK2β and the tetrameric α2β2 CK2 holoenzyme. Even if the structural knowledge of CK2 is very extended, no high resolution 3D-structure are available for the C-terminal part of CK2α, which has been deleted for the crystallization purpose. Moreover the structure of the CK2α’ has been solved but no structural information are present for the tetrameric holoenzyme with this catalytic subunit.
To address this issue, one part of my PhD project focused on the production and the structural characterization of the full-length wild type CK2α and a phosphomimetic mutant in the tetrameric holoenzyme, in order to study the possible structural role of the C-terminus. Starting from the three holoenzyme structures solved we were able to determine some new holoenzyme structural features, in particular the new interface of interaction between the subunits within the tetramer and the so far unknown symmetry of the complex. Moreover we dealt with the development of a purification protocol of the CK2α’2β2 holoenzyme (and a chimeric CK2αα’β2) in quantities appropriate for structural approaches.
The second part of the PhD focused on the structural characterization of a new potent dual inhibitor K164 which is specific for CK2 and Pim1; the crystal structure of the inhibitor in complex with the CK2α336 has been solved at 1.25 Å, which is the highest resolution ever reached for CK2.Il soggetto di questa tesi è la protein chinasi CK2, una famiglia di enzimi che negli uomini è composta da due subunità catalitiche, CK2α e CK2α’, e da una subunità regolatoria, CK2β. CK2 è una Ser/Thr protein chinasi acidofila altamente conservata nel mondo eucariote, presente in differenti compartimenti cellulari. CK2 è un membro della superfamiglia delle protein chinasi eucariotiche (EPKs), con la subunità catalitica correlata mediante omologia di sequenza e caratteristiche strutturali alle altre 478 chinasi della superfamiglia.
CK2 mostra alcune caratteristiche singolari, come la sua elevata attività costitutiva e la mancanza di un importante meccanismo di regolazione, il quale rende CK2 unica rispetto alle altre chinasi. Con centinaia di substrati, CK2 è coinvolta in numerosi processi biologici, risultando essenziale per la vitalità cellulare: il knockout del gene CK2β nel modello murino è letale anche a livello di singola cellula, il knockout del gene CK2α è letale al giorno 10.5 dello sviluppo embrionale e il knockout CK2α' (espresso solo nel cervello e testicoli) in topo è vitale con alcuni difetti di spermatogenesi. CK2 partecipa alla progressione del ciclo cellulare, all'espressione genica, alla crescita cellulare e alla differenziazione e all’embriogenesi. Down-regulation di CK2 porta all'apoptosi cellulare mentre una sovra-attivazione anomala è stata trovata accoppiata a diverse malattie: la rilevanza clinica di CK2 risiede nel fatto che alti livelli di attività della proteina sono stati trovati in diversi tipi di tumori, come alla testa e al collo, ai reni, al seno, alla prostata e al polmone.
Un ampio spettro di inibitori di CK2, permeabili alle cellule e specifici per il sito dell’ATP, sono attualmente disponibili e si stanno rivelando utili per analizzare le funzioni biologiche della proteina; queste piccole molecole sono in grado di indurre l'apoptosi delle cellule tumorali senza alcun effetto analogo sulle loro controparti "normali". Uno di questi inibitori, CX-4945, è recentemente entrato in studi clinici per il trattamento di tumori solidi avanzati, malattia di Castelman e mieloma multiplo.
CK2 è considerato un enzima costitutivamente attivo e, a differenza di molte altre protein chinasi, non richiede fosforilazione per l'attivazione. Il meccanismo di regolazione di CK2 non è stato ancora stabilito, tuttavia è chiaro che si differenzia da quelli comunemente utilizzati dalle altre protein chinasi.
Decine di strutture cristallografiche di CK2 sono state risolte e hanno evidenziato le caratteristiche strutturali delle principali entità di CK2: la subunità catalitica CK2α, la subunità regolatoria CK2β e l’oloenzima tetramerico CK2α2β2. Anche se la conoscenza strutturale di CK2 è molto estesa, non è disponibile alcuna struttura 3D a elevata risoluzione per la parte C-terminale di CK2α, che è sempre stata deleta per scopi di cristallizzazione. Inoltre, malgrado la struttura di CK2α' sia stata risolta, non sono presenti alcune informazioni strutturali per l’oloenzima tetramerico con questa subunità catalitica.
Per raggiungere questo obiettivo, una parte del mio progetto di dottorato si è focalizzata sulla produzione e sulla caratterizzazione strutturale di CK2α wild type (completa della parte C-terminale) e di un mutante fosfomimetico nell’oloenzima tetramerico, al fine di studiare il possibile ruolo strutturale del C-terminale. Partendo da tre strutture dell’oloenzima risolte abbiamo potuto determinare alcune nuove caratteristiche strutturali dell’oloenzima, in particolare la nuova interfaccia di interazione tra le subunità all'interno del tetramero e la simmetria del complesso, finora sconosciuta. Inoltre ci siamo occupati dello sviluppo di un protocollo di purificazione dell’oloenzima CK2α’2β2 (e di una forma chimerica CK2αα’β2) in quantità appropriate per approcci strutturali.
La seconda parte del dottorato si è focalizzata sulla caratterizzazione strutturale del complesso con un nuovo potente inibitore duale (K164) il quale è specifico per CK2 e Pim1; la struttura cristallina di CK2α336 in complesso con l’inibitore è stata risolta a 1.25 Å, che è la più alta risoluzione mai raggiunta per CK2
Active Form of the Protein Kinase CK2 alpha(2)beta(2) holoenzyme Is a Strong Complex with Symmetric Architecture
No full textCK2 is a protein kinase essential for cell viability whose activity is altered in several cancers. Its mechanisms of regulation differ from those common to other eukaryotic protein kinases and are not entirely established yet. Here we present crystal structures of the monomeric form of the alpha(2)beta(2) holoenzyme that allow refining a formerly proposed structural model for activity regulation by oligomerization. Previous crystal structures of the CK2 holoenzyme show an asymmetric arrangement of the two alpha catalytic subunits around the obligate beta(2) regulatory subunits. Asymmetric alpha(2)beta(2) tetramers are organized in trimeric rings that correspond to inactive forms of the enzyme. The new crystal structures presented here reveal the symmetric architecture of the isolated active tetramers. The dimension and the nature of the alpha/beta interfaces configure the holoenzyme as a strong complex that does not spontaneously dissociate in solution, in accordance with the low dissociation constant (similar to 4 nM)
Structural and functional analysis of the flexible regions of the catalytic α-subunit of protein kinase CK2.
No full textCK2 is a Ser/Thr protein kinase essential for cell viability. Its activity is anomalously high in several solid (prostate, mammary gland, lung, kidney and head and neck) and haematological tumours (AML, CML and PML), creating conditions favouring the onset of cancer. Cancer cells become addicted to high levels of CK2 activity and therefore this kinase is a remarkable example of "non-oncogene addiction". CK2 is a validated target for cancer therapy with one inhibitor in phase I clinical trials. Several crystal structures of CK2 are available, many in complex with ATP-competitive inhibitors, showing the presence of regions with remarkable flexibility. We present the structural characterisation of these regions by means of seven new crystal structures, in the apo form and in complex with inhibitors. We confirm previous findings about the unique flexibility of the CK2alpha catalytic subunit in the hinge/alphaD region, the p-loop and the beta4beta5 loop, and show here that there is no clear-cut correlation between the conformations of these flexible zones. Our findings challenge some of the current interpretations on the functional role of these regions and dispute the hypothesis that small ligands stabilize an inactive state. The mobility of the hinge/alphaD region in the human enzyme is unique among protein kinases, and this can be exploited for the development of more selective ATP-competitive inhibitors. The identification of different ligand binding modes to a secondary site can provide hints for the design of non-ATP-competitive inhibitors targeting the interaction between the alpha catalytic and the beta regulatory subunits
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
- …
