Residues at the Active Site of the Esterase 2 fromAlicyclobacillus acidocaldarius Involved in Substrate Specificity and Catalytic Activity at High Temperature

The recently solved three-dimensional structure of the thermophilic esterase 2 from Alicyclobacillus acidocaldarius allowed us to have a snapshot of an enzyme-sulfonate complex, which mimics the second stage of the catalytic reaction, namely the covalent acyl-enzyme intermediate. The aim of this work was to design, by structure-aided analysis and to generate by site-directed and saturation mutagenesis, EST2 variants with changed substrate specificity in the direction of preference for monoacylesters whose acyl-chain length is greater than eight carbon atoms. Positions 211 and 215 of the polypeptide chain were chosen to introduce mutations. Among five variants with single and double amino acid substitutions, three were obtained, M211S, R215L, and M211S/R215L, that changed the catalytic efficiency profile in the desired direction. Kinetic characterization of mutants and wild type showed that this change was achieved by an increase in k(cat) and a decrease in K(m) values with respect to the parental enzyme. The M211S/R215L specificity constant for p-nitrophenyl decanoate substrate was 6-fold higher than the wild type. However, variants M211T, M211S, and M211V showed strikingly increased activity as well as maximal activity with monoacylesters with four carbon atoms in the acyl chain, compared with the wild type. In the case of mutant M211T, the k(cat) for p-nitrophenyl butanoate was 2.4-fold higher. Overall, depending on the variant and on the substrate, we observed improved catalytic activity at 70 degrees C with respect to the wild type, which was a somewhat unexpected result for an enzyme with already high k(cat) values at high temperature. In addition, variants with altered specificity toward the acyl-chain length were obtained. The results were interpreted in the context of the EST2 three-dimensional structure and a proposed catalytic mechanism in which k(cat), e.g. the limiting step of the reaction, was dependent on the acyl chain length of the ester substrate

A Novel Aspartyl Proteinase from Apocrine Epithelia and Breast Tumors

Abstract GCDFP-15 (gross cysticdisease fluid protein,15 kDa) is a secretory marker of apocrine differentiation in breast carcinoma. In human breast cancer cell lines, gene expression is regulated by hormones, including androgens and prolactin. The protein is also known under different names in different body fluids such as gp17 in seminal plasma. GCDFP-15/gp17 is a ligand of CD4 and is a potent inhibitor of T-cell apoptosis induced by sequential CD4/T-cell receptor triggering. We now report that GCDFP-15/gp17 is a protease exhibiting structural properties relating it to the aspartyl proteinase superfamily. Unexpectedly, GCDFP-15/gp17 appears to be related to the retroviral members rather than to the known cellular members of this class. Site-specific mutagenesis of Asp22 (predicted to be catalytically important for the active site) and pepstatin A inhibition confirmed that the protein is an aspartic-type protease. We also show that, among the substrates tested, GCDFP-15/gp17 is specific for fibronectin. The study of GCDFP-15/gp17-mediated proteolysis may provide a handle to understand phenomena as diverse as mammary tumor progression and fertilization

A Cloud Architecture for Managing IoT-aware Applications According to Knowledge Processing Rules

The Web of Things paradigm has represented a shift in the conjunction of the Internet of Things (IoT) with people, as it allows treating a smart object as a Web resource. While in a first phase the challenge was the physically management of smart objects, the current demand is to help users in profitably introducing IoT in their own daily life.The paper presents a software architecture for IoT systems able to manage the behaviour of involved IoT entities basing on knowledge processing tools. The main goal is informing the user of the occurrence of events of interest semantically determined starting from actual state of the environment. The architecture exploits the potentialities of the Web of Topics (WoX) approach, a conceptual model that simplifies the designing of IoT applications. Leveraging the WoX approach, the architecture introduces an innovative way to mine knowledge from IoT devices aside from any technological background, so that facing the intrinsic heterogeneity affecting IoT entities. The discussed architecture is composed by different modules integrated into an Enterprise Service Bus (ESB), strongly decoupled and provided with RESTful-compliant web interfaces to communicate each other and with the external environment, according to a SOA structure. The paper shows how the system is able to receive data coming from sensors and to semantically interpret them by means of a series of business rules that act as knowledge processor

The Aes protein and the monomeric alpha-galactosidase from Escherichia coli form a non-covalent complex. Implications for the regulation of carbohydrate metabolism.

Aes, a 36-kDa acetylesterase fromEscherichia coli, belongs to the hormone-sensitive lipase family, and it is involved in the regulation of MalT, the transcriptional activator of the maltose regulon. The activity of MalT is depressed through a direct protein-protein interaction with Aes. Although the effect is clear-cut, the meaning of this interaction and the conditions that trigger it still remain elusive. To perform a comparative thermodynamic study between the mesophilic Aes protein and two homologous thermostable enzymes, Aes was overexpressed in E. coli and purified. At the last step of the purification procedure the enzyme was eluted from a Mono Q HR 5/5 column as a major form migrating, anomalously, at 56 kDa on a calibrated Superdex 75 column. A minor peak that contains the Aes protein and a polypeptide of 50 kDa was also detected. By a combined analysis of size-exclusion chromatography and surface-enhanced laser desorption ionization-time of flight mass spectrometry, it was possible to demonstrate the presence in this peak of a stable 87-kDa complex, containing the Aes protein itself and the 50-kDa polypeptide in a 1:1 ratio. The homodimeric molecular species of Aes and of the 50-kDa polypeptide were also detected. The esterase activity associated with the 87-kDa complex, when assayed with p-nitrophenyl butanoate as substrate, proved 6-fold higher than the activity of the major Aes form of 56 kDa. Amino-terminal sequencing highlighted that the 50-kDa partner of Aes in the complex was the α-galactosidase from E. coli. TheE. coli cells harboring plasmid pT7-SCII-aesand, therefore, expressing Aes were hampered in their growth on a minimal medium containing raffinose as a sole carbon source. Because α-galactosidase is involved in the metabolism of raffinose, the above findings suggest a potential role of Aes in the regulation of carbohydrate metabolism in E. coli

gis ila the gis for italian logistics in antarctica

A geographic information system (GIS) is a hardware/software tool which is very effective in collecting, storing, searching, handling, and visualizing geographic data together with their descriptive attributes. Such a tool is very helpful to approach a wide range of situations, including many logistic problems. ENEA, as the agency having the task of implementing the Italian Antarctic Program (PNRA), has decided to use a GIS, in order to increase the efficiency in managing the huge amount of data collected in the course of Italian activity in Antarctica, which counts fifteen expeditions up to now

SSo

Previously, we reported from the Sulfolobus solfataricus open reading frame (ORF) SSO2517 the cloning, overexpression and characterization of an esterase belonging to the hormone-sensitive lipase (HSL) family and apparently having a deletion at the N-terminus, which we named SsoNΔ. Searching the recently reported Sulfolobus acidocaldarius genome by sequence alignment, using SSO2517 as a query, allowed identity of a putative esterase (ORF SAC1105) sharing high sequence similarity (82%) with SSO2517. This esterase displays an N-terminus and total length similar to other known esterases of the HSL family. Analysis of the upstream DNA sequence of SS02517 revealed the possibility of expressing a longer version of the protein with an extended N-terminus; however, no clear translation signal consistent with a longer protein version was detected. This new version of SSO2517 was cloned, over-expressed, purified and characterized. The resulting protein, named SsoNΔlong, was 15-fold more active with the substrate p-nitrophenyl hexanoate than SsoNΔ. Furthermore, SsoNΔlong and SsoNΔ displayed different substrate specificities for triacylglycerols. These results and the phylogenetic relationship between S. solfataricus and S. acidocaldarius suggest a common origin of SSO2517 and SAC1105 from an ancestral gene, followed by divergent evolution. Alternatively, a yet-to-be discovered mechanism of translation that directs the expression of SsoNΔlong under specific metabolic conditions could be hypothesized

A Substrate-induced Switch in the Reaction Mechanism of a Thermophilic Esterase KINETIC EVIDENCES AND STRUCTURAL BASIS

The reaction mechanism of the esterase 2 (EST2) from Alicyclobacillus acidocaldarius was studied at the kinetic and structural level to shed light on the mechanism of activity and substrate specificity increase previously observed in its double mutant M211S/R215L. In particular, the values of kinetic constants (k1, k(-1), k2, and k3) along with activation energies (E1, E(-1), E2, and E3) were measured for wild type and mutant enzyme. The previously suggested substrate-induced switch in the reaction mechanism from kcat=k3 with a short acyl chain substrate (p-nitrophenyl hexanoate) to kcat=k2 with a long acyl chain substrate (p-nitrophenyl dodecanoate) was validated. The inhibition afforded by an irreversible inhibitor (1-hexadecanesulfonyl chloride), structurally related to p-nitrophenyl dodecanoate, was studied by kinetic analysis. Moreover the three-dimensional structure of the double mutant bound to this inhibitor was determined, providing essential information on the enzyme mechanism. In fact, structural analysis explained the observed substrate-induced switch because of an inversion in the binding mode of the long acyl chain derivatives with respect to the acyl- and alcohol-binding sites

Top-Down Delivery of IoT-based Applications for Seniors Behavior Change Capturing Exploiting a Model-Driven Approach

Developing Internet of Things (IoT) requires expertise and considerable skills in different fields in order to cover all the involved heterogeneous technologies, communication formats and protocols. Developers and experts ask for new solutions that speed up the prototyping of IoT applications. One of these solutions is Web of Topics (WoX) middleware, a model-driven Cloud platform that aims to ease IoT applications developing, introducing a strong semantic abstraction of the IoT concepts. In WoX, almost all the IoT entities and concepts are limited to the concept of Topic, i.e. an entity containing the value of a feature of interest that we intend to detect. The local counterpart of WoX is L-WoX (Local-Web of Topics), which manages local instances of features of interest, allowing mobile applications to collaborate among them, offering and receiving data to/from smart objects, and enabling the communication with WoX Cloud platform. The presented study leverages WoX approach for showing an experience in rapid design and prototyping of an ambient assisted living system that detects the movements of elderly persons in their home, acquiring data through sensors in an unobtrusive way. Moreover, The paper shows that the chosen model-driven solution is very suitable in a top-down approach, starting from users requirements: the created system simplifies the user-centered design of IoT applications, adopting a full top-down approach from user required to the technological solution

Relazione tecnica sulle attività della campagna oceanografica “Evatir 2017”

A partire dal 2017, la campagna nelle GSA 9 e 10 è parte integrante del Piano di Lavoro Nazionale Raccolta Dati Alieutici 2017-2019 ai sensi della Council Regulation (EC) N° 199/2008, Commission Regulation (EC) N°665/2008 and Commission Implementing Decision (EU) 2016/1251. La presente relazione tecnica riporta l’attività svolta nella Campagna oceanografica “EVATIR 2017”, nell’estate del 2017 a bordo della N/O Dallaporta,bdurante i survey acustici realizzati nello Stretto di Sicilia (GSA 16), nel Mar Tirreno centrale e meridionale (GSA 10), nel Mar Tirreno settentrionale e nel Mar Ligure (GSA 9) nell’ambito del modulo MEDIAS del Piano Nazionale PLNRDA 2017-2019. Secondo il protocollo MEDIAS (MEDIAS, 2017), insieme all’acquisizione dei dati acustici sulla piattaforma continentale, sono state eseguite stazioni di pesca sui piccoli pelagici con rete volante provvista di sensori per il controllo dell’apertura e della posizione della rete nella colonna d’acqua, e rilevamenti con sonda multiparametrica CTD in stazioni predeterminate lungo i transetti del survey

Preface of the 31st Italian Symposium on Advanced Database Systems

This volume contains the proceedings of the 31st Italian Symposium on Advanced Database Systems (SEBD - Sistemi Evoluti per Basi di Dati), held in Galzinagno Terme (Padua, Italy) from 2 to 5 July 2023.</p