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Molecular Mechanisms of Recognition of Coagulation Factors (Meccanismi Molecolari di Riconoscimento dei Fattori della Coagulazione)
No full textThe biochemical pathways sustaining living organisms, traditionally regarded as independently organized systems, are actually extensively connected by non-canonical protein interactions. The disruption of this finely regulated homeostasis, largely yet to unravel, results in various pathological manifestations.
Haemostasis (Chapter 1) is a defence response triggering after vessel walls injuries, which is articulated in the cascade activation of the blood coagulation factors, resulting in the generation of a localized clot. The central reaction of the coagulation cascade is prothrombin activation to mature a-thrombin by FXa, through the generation of the physiologically relevant intermediate prethrombin-2. Active a-thrombin is an ellipsoidal protein, composed of two six-stranded b-barrels encompassing, at their interface, the catalytic triad (His57, Asp102, Ser195). Opposite to the negatively charged active site, two extra positive regions of binding, named exosite I and exosite II, mediate the recognition with several physiologic ligands and substrates. Mature a-thrombin plays a pivotal role in haemostasis, entailing both procoagulant functions (platelets aggregation, fibrin generation) and an anticoagulant one (protein C activation). Beyond coagulation, this serine protease acts at the interface between inflammation, cellular proliferation, and neurodegenerative diseases.
The interplay between a-thrombin and proteins traditionally belonging to the central nervous system is undoubtedly a pioneering and yet unexplored topic (Chapter 2.1). When present at high cerebral concentrations a-thrombin triggers a pathologic pro-inflammatory state in the brain, which may be involved in the onset of neurodegenerative diseases. In detail, we refer to as synucleinopathies for a branch of diseases (i.e. Parkinson’s disease) in which intracellular proteinaceous aggregates, mainly composed of a-synuclein, localize both in neurons and in glia. a-synuclein is an abundant presynaptic protein, belonging to the family of naturally unfolded proteins (NUPs), whose physiologic function is still matter of debate. Strikingly, beyond central nervous system, a-synuclein has been detected in plasma and in the haematopoietic lineage, particularly in platelets. Surprisingly, patients suffering from Parkinson’s disease, featuring high a-synuclein concentrations, are characterized by lower ischaemic attacks, due to platelets abnormalities and impaired aggregation. Being these cells the main trigger of a-thrombin, we purposed to investigate the interaction between this enzyme and a-synuclein (Chapter 2.2). Our data clearly demonstrate that the two proteins bind with an affinity physiologically relevant for the concentrated microenvironment surrounding platelets. In the binary complex, a-synuclein interacts promiscuously with a-thrombin exosites by its negatively charged de-structured C-terminus, thus scavenging hyper-aggregation phaenomena.
In this intricate network, positive upregulation between coagulation and inflammation has been well established and extensively studied (Chapter 3.1). During sepsis, systematically activated immune response results in an exaggerated and detrimental inflammation, usually coupled to disseminated intravascular coagulation. In this scenario, exogenous proteases may play a relevant role during the early stages of the infection by directly activating coagulation. Several pathogen microorganisms express and secrete subtilisin-like serine proteases (subtilases), characterized by a broad specificity of cleavage. We purposed to investigate the effects of subtilases-catalysed proteolysis of thrombin zymogens prethrombin-2 (Chapter 3.2) and prothrombin (Chapter 3.3) by using the commercially available subtilisin Carlsberg, as a prototype for the superfamily. From the proteolysis and enzymatic assays data, it strikingly emerged that subtilisin activates both the zymogens to a novel thrombin-like specie, by the non-canonical hydrolysis of Ala470(149a)-Asn471(149b) peptide bond. The novel active specie, we named sPre2, is a non-covalent complex featuring the same a-thrombin cleavage specificity, with a catalytic efficiency ≈150-fold inferior to the physiologic enzyme. From fluorescence titrations and surface binding resonance, it emerged that sPre2 is characterized by a fully competent exosite II, an imperfect exosite I and a correctly moulded active site, which however features an impaired mechanism of substrate conversion.
Both experimental and clinical evidences clearly demonstrate the crucial importance of maintaining coagulation homeostasis, which, in physiologic conditions, is slightly unbalanced towards a haemorrhagic state to keep blood fluid in the intact vessels. A disruption of this equilibrium due to increase of functioning leads to pathological manifestations generically referred to as thrombosis (Chapter 4.1). Unfortunately, classical anticoagulant therapy presents well-documented limitations and bleeding side effects, driving the continuous efforts to develop new, safer drugs. In the late years, a hot research topic is represented by the engineering of natural anticoagulants from hematophagous organisms. Among all, anticoagulant hirudin from the medicinal leeches is the most popular compound, being a-thrombin most potent and specific natural inhibitor. In this work, we propose a novel strategy for hirudin production in E. coli, by conjugation to SUMO (small ubiquitin-like modifier protein), a eukaryotic chaperon which enhances protein folding and solubility (Chapter 4.2). The so-obtained recombinant hirudin is characterized by the same folding, spectroscopic features, and anti-thrombin activity of the natural variant.
In conclusion, coagulation is undoubtedly one of the most articulated and fascinating physio-pathologic systems regulating body homeostasis, displaying several connections with other biochemical pathways. The molecular mechanisms of recognition of the coagulation factors, yielding traditional or original protein interactions, is still a highly unexplored topic
Noncoded amino acids in protein engineering: Structure-activity relationship studies of hirudin-thrombin interaction
Full text linkThe advent of recombinant DNA technology allowed to site-specifically insert, delete, or mutate almost any amino acid in a given protein, significantly improving our knowledge of protein structure, stability, and function. Nevertheless, a quantitative description of the physical and chemical basis that makes a polypeptide chain to efficiently fold into a stable and functionally active conformation is still elusive. This mainly originates from the fact that nature combined, in a yet unknown manner, different properties (i.e., hydrophobicity, conformational propensity, polarizability, and hydrogen bonding capability) into the 20 standard natural amino acids, thus making difficult, if not impossible, to univocally relate the change in protein stability or function to the alteration of physicochemical properties caused by amino acid exchange(s). In this view, incorporation of noncoded amino acids with tailored side chains, allowing to finely tune the structure at a protein site, would facilitate to dissect the effects of a given mutation in terms of one or a few physicochemical properties, thus much expanding the scope of physical organic chemistry in the study of proteins. In this review, relevant applications from our laboratory will be presented on the use of noncoded amino acids in structure-activity relationships studies of hirudin binding to thrombin
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
Non-canonical proteolytic activation of human prothrombin by subtilisin from Bacillus subtilis may shift the procoagulantâ anticoagulant equilibrium toward thrombosis
Full text linkBlood coagulation is a finely regulated physiological process culminating with the factor Xa (FXa)-mediated conversion of the prothrombin (ProT) zymogen to active -thrombin (T). In the prothrombinase complex on the platelet surface, FXa cleaves ProT at Arg-271, generating the inactive precursor pre-thrombin-2 (Pre2), which is further attacked at Arg-320 –Ile-321 to yield mature T. Whereas the mechanism of physiological ProT activation has been elucidated in great detail, little is known about the role of bacterial proteases, possibly released in the bloodstream during infection, in inducing blood coagulation by direct proteolytic ProT activation. This knowledge gap is particularly concerning, as bacterial infections are frequently complicated by severe coagulopathies. Here, we show that addition of subtilisin (50 nM to 2 M), a serine protease secreted by the non-pathogenic bacterium Bacillus subtilis, induces plasma clotting by proteolytically converting ProT into active Pre2, a nicked Pre2 derivative with a single cleaved Ala-470 –Asn-471 bond. Notably, we found that this non-canonical cleavage at Ala-470 –Asn-471 is instrumental for the onset of catalysis in Pre2, which was, however, reduced about 100 –200-fold compared with T. Of note, Pre2 could generate fibrin clots from fibrinogen, either in solution or in blood plasma, and could aggregate human platelets, either isolated or in whole blood. Our findings demonstrate that alternative cleavage of ProT by proteases, even by those secreted by non-virulent bacteria such as B. subtilis, can shift the delicate procoagulant–anticoagulant equilibrium toward thrombosis
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
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