105 research outputs found
Biochemical and structural characterization of proteins involved in prokaryotic DNA metabolism as source of novel drug targets and biotechnological solutions
No full textThe aim of the presented PhD project was the biochemical and structural characterization of proteins and enzymes involved in prokaryotic DNA metabolism as potential target for antitubercular drug discovery as well as for the design of biotechnological tools for application in molecular biology. A variant of the 06-DNA-alkyl-guanine-DNA-alkyl-transferases (OGT) from Saccharolobus solfataricus (HS) has been proposed as a thermostable self-labelling protein tag in order to expand this technology to harsh experimental conditions and extreme environments. We present here the crystal structure of H5 protein in complex with the fluorescent probe functional to a structure based protein engineering aiming at further evolution of the proposed technology based on a chemo-enzymatic approach for the labelling reaction. The second part of the presented thesis focused on the characterization of protein complexes from Mycobacterium tuberculosis (MTB)
involved in the Nucleotide Excision Repair (NER) pathway and the initiation phase of DNA replication. To this aim we obtained a low-resolution model of the UvrA-UvrB complex acting in the damage recognition step along NER pathway. The structural model allow us to map three residues involved in protein-protein interaction whose key role was further confirmed by site directed mutagenesis experiments. On the other hands, we obtained preliminary results on the biochemical characterization of a novel helicase loader acting during DNA replication in MTB, namely DciA. In order to shed light on mechanistic aspects of DciA-dependent helicase in MTB, we investigated the secondary structure element of DciA domains protein using limited proteolysis coupled with native mass spectrometry. Moreover, we determined the kinetic constants of the interaction between DciA loader and the target helicase by means of Surface Plasmon Resonance
Targeting Genome Integrity in Mycobacterium Tuberculosis: From Nucleotide Synthesis to DNA Replication and Repair
No full textMycobacterium tuberculosis (MTB) is the causative agent of tuberculosis (TB), an ancient disease which still today causes 1.4 million deaths worldwide per year. Long-term, multi-agent anti-tubercular regimens can lead to the anticipated non-compliance of the patient and increased drug toxicity, which in turn can contribute to the emergence of drug-resistant MTB strains that are not susceptible to first- and second-line available drugs. Hence, there is an urgent need for innovative antitubercular drugs and vaccines. A number of biochemical processes are required to maintain the correct homeostasis of DNA metabolism in all organisms. Here we focused on reviewing our current knowledge and understanding of biochemical and structural aspects of relevance for drug discovery, for some such processes in MTB, and particularly DNA synthesis, synthesis of its nucleotide precursors, and processes that guarantee DNA integrity and genome stability. Overall, the area of drug discovery in DNA metabolism appears very much alive, rich of investigations and promising with respect to new antitubercular drug candidates. However, the complexity of molecular events that occur in DNA metabolic processes requires an accurate characterization of mechanistic details in order to avoid major flaws, and therefore the failure, of drug discovery approaches targeting genome integrity
Non-SUMOylated alternative spliced isoforms of alpha-synuclein are more aggregation-prone and toxic
No full textCrystal structure of a thermophilic O6-alkylguanine-DNA alkyltransferase-derived self-labeling protein-tag in covalent complex with a fluorescent probe
No full textThe self-labeling protein tags are robust and versatile tools for studying different molecular aspects of cell biology. In order to be suitable for a wide spectrum of experimental conditions, it is mandatory that these systems are stable after the fluorescent labeling reaction and do not alter the properties of the fusion partner. SsOGT-H5 is an engineered variant alkylguanine-DNA-alkyl-transferase (OGT) of the hyperthermophilic archaeon Sulfolobus solfataricus, and it represents an alternative solution to the SNAP-tag® technology under harsh reaction conditions. Here we present the crystal structure of SsOGT-H5 in complex with the fluorescent probe SNAP-Vista Green® (SsOGT-H5-SVG) that reveals the conformation adopted by the protein upon the trans-alkylation reaction with the substrate, which is observed covalently bound to the catalytic cysteine residue. Moreover, we identify the amino acids that contribute to both the overall protein stability in the post-reaction state and the coordination of the fluorescent moiety stretching-out from the protein active site. We gained new insights in the conformational changes possibly occurring to the OGT proteins upon reaction with modified guanine base bearing bulky adducts; indeed, our structural analysis reveals an unprecedented conformation of the active site loop that is likely to trigger protein destabilization and consequent degradation. Interestingly, the SVG moiety plays a key role in restoring the interaction between the N- and C-terminal domains of the protein that is lost following the new conformation adopted by the active site loop in the SsOGT-H5-SVG structure. Molecular dynamics simulations provide further information into the dynamics of SsOGT-H5-SVG structure, highlighting the role of the fluorescent ligand in keeping the protein stable after the trans-alkylation reaction
Universalität als Werthaftigkeit. Das „Scheitern“ des Fichtebuchs von Emil Lask
No full textThe author analyzes Emil Lask’s Fichtes Idealismus und
die Geschichte (1902), remarking its failure in the attempt of founding an integrative
relationship between value and real individuality, and of reconciling the transcendence of
validity with the individual foundation of value. The possibility of conceiving an integrative
dimension between value and reality supposes the rejection of any valuable universalism
and the acknowledgement of all the relativizing consequences deriving from the fact that
value emerges only within the historical individuality. Anyway, the author believes that an
universality of value, understood as valuableness, that is the common capacity to attribute
value which is typical of the historical subjects, is still conceivable
Begriff und Wirklichkeit. Emil Lask und die Frage des Emanantismus
No full textThe essay shows how the anti-emanationism represents a central element in Emil Lask’s thought. The author focuses especially on Lask’s methodological and social-juridical reflections, referring broadly to Lask’s correspondence. Lask’s concept of knowledge is viewed as a concept constantly suspended between the rejection of emanationism and the necessity of integrating the analytical logic
La storia è il risultato della storiografia?
No full textStarting from the last book of Fulvio Tessitore (A partire da Dilthey. Trittico anti-hegeliano: Weber, Meinecke, Rosenzweig, Roma, 2013) the author discusses the central points of Tessitorian interpretation of historicism. In particular the author argues the idea of historicism intended as a complete and radical negation of any form of ontology. The author is convinced that the historicistical anti-ontologism is equivalent to the affirmation of the historical reality in its autonomy from any absolute and its constitutive connection with human action
La genesi della Kulturphilosophie
No full textThe author reconstructs the process of the genesis of the modern philosophy of culture, analysing the positions of Vico, Rousseau, Kant, Herder. The author focuses on the two essential theoretical components of this process, which are the cultural critique and the “Copernican” principle
Max Weber e il dibattito attuale sulle migrazioni
No full textMax Weber and the Current Debate on Migration. Drawing on Max Weber’s famous Antrittsvorlesung (1895), the author develops an ideal type framework to categorize various perspectives on the migration issue. Based on two contrasting attitudes – the axiological-differential and the universal-relativistic – the author critically examines the positions of Thilo Sarrazin, Bassam Tibi, and Aladdin El-Mafaalani
Il concetto di cultura fra epocalità e autotrasparenza. Note per una Kulturphilosophie storica
No full textThe concept of culture between epochality and self-trasparency. Some remarks for a historical Kulturphilosophie. The author dwells on Ralf Konersmann’s positions on the philosophy of culture. He emphasizes the epochal structure of the concept of culture and connects it to the defective structure of modern culture. The author rejects any teleological construction of the origins of the philosophy of culture and reflects on the concept of self-transparency of culture, interpreting it as a specific way in which culture gives itself in the modern era
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