Ivan Mičetić

Biochemical Characterization of Highly Purified Leucine-Rich Repeat Kinases 1 and 2 Demonstrates Formation of Homodimers

Leucine-rich repeat kinase 1 and 2 (LRRK1 and LRRK2) are large multidomain proteins containing kinase, GTPase and multiple protein-protein interaction domains, but only mutations in LRRK2 are linked to familial Parkinsons disease (PD). Independent studies suggest that LRRK2 exists in the cell as a complex compatible with the size of a dimer. However, whether this complex is truly a homodimer or a heterologous complex formed by monomeric LRRK2 with other proteins has not been definitively proven due to the limitations in obtaining highly pure proteins suitable for structural characterization. Here, we used stable expression of LRRK1 and LRRK2 in HEK293T cell lines to produce recombinant LRRK1 and LRRK2 proteins of greater than 90% purity. Both purified LRRKs are folded, with a predominantly alpha-helical secondary structure and are capable of binding GTP with similar affinity. Furthermore, recombinant LRRK2 exhibits robust autophosphorylation activity, phosphorylation of model peptides in vitro and ATP binding. In contrast, LRRK1 does not display significant autophosphorylation activity and fails to phosphorylate LRRK2 model substrates, although it does bind ATP. Using these biochemically validated proteins, we show that LRRK1 and LRRK2 are capable of forming homodimers as shown by single-particle transmission electron microscopy and immunogold labeling. These LRRK dimers display an elongated conformation with a mean particle size of 145 Å and 175 Å respectively, which is disrupted by addition of 6M guanidinium chloride. Immunogold staining revealed double-labeled particles also in the pathological LRRK2 mutant G2019S and artificial mutants disrupting GTPase and kinase activities, suggesting that point mutations do not hinder the dimeric conformation. Overall, our findings indicate for the first time that purified and active LRRK1 and LRRK2 can form dimers in their full-length conformation.

Antibacterial activity of silver nanoparticles: sensitivity of different Salmonella serovars.

Salmonella spp. is one of the main causes of foodborne illnesses in humans worldwide. Consequently, great interest exists in reducing its impact on human health by lowering its prevalence in the food chain. Antimicrobial formulations in the form of nanoparticles exert bactericidal action due to their enhanced reactivity resultant from their high surface/volume ratio. Silver nanoparticles (AgNPs) are known to be highly toxic to Gram-negative and Gram-positive microorganisms, including multidrug resistant bacteria. However, few data concerning their success against different Salmonella serovars are available. Aims of the present study were to test the antimicrobial effectiveness of AgNPs, against Salmonella Enteritidis, Hadar, and Senftenberg, and to investigate the causes of their different survival abilities from a molecular point of view. Results showed an immediate, time-limited and serovar-dependent reduction of bacterial viability. In the case of S. Senftenberg, the reduction in numbers was observed for up to 4 h of incubation in the presence of 200 mg/l of AgNPs; on the contrary, S. Enteritidis and S. Hadar resulted to be inhibited for up to 48 h. Reverse transcription and polymerase chain reaction experiments demonstrated the constitutive expression of the plasmidic silver resistance determinant (SilB) by S. Senftenberg, thus suggesting the importance of a cautious use of AgNPs.

Phytotoxicity of ionic, micro- and nano-sized iron in three plant species

Potential environmental impacts of engineered nanoparticles (ENPs) can be understood taking into consideration phytotoxicity. We reported on the effects of ionic (FeCl3), micro- and nano-sized zerovalent iron (nZVI) about the development of three macrophytes: Lepidium sativum, Sinapis alba and Sorghum saccharatum. Four toxicity indicators (seed germination, seedling elongation, germination index and biomass) were assessed following exposure to each iron concentration interval: 1.29-1570mg/L (FeCl3), 1.71-10.78mg/L (micro-sized iron) and 4.81-33,560mg/L (nano-iron). Exposure effects were also observed by optical and transmission electron microscopy. Results showed that no significant phytotoxicity effects could be detected for both micro- and nano-sized zerovalent irons, including field nanoremediation concentrations. Biostimulation effects such as an increased seedling length and biomass production were detected at the highest exposure concentrations. Ionic iron showed slight toxicity effects only at 1570mg/L and, therefore, no median effect concentrations were determined. By microscopy, ENPs were not found in palisade cells or xylem. Apparently, aggregates of nZVI were found inside S. alba and S. saccharatum, although false positives during sample preparation cannot be excluded. Macroscopically, black spots and coatings were detected on roots of all species especially at the most concentrated treatments.

Tools and data services registry: a community effort to document bioinformatics resources

Life sciences are yielding huge data sets that underpin scientific discoveries fundamental to improvement in human health, agriculture and the environment. In support of these discoveries, a plethora of databases and tools are deployed, in technically complex and diverse implementations, across a spectrum of scientific disciplines. The corpus of documentation of these resources is fragmented across the Web, with much redundancy, and has lacked a common standard of information. The outcome is that scientists must often struggle to find, understand, compare and use the best resources for the task at hand. Here we present a community-driven curation effort, supported by ELIXIR—the European infrastructure for biological information—that aspires to a comprehensive and consistent registry of information about bioinformatics resources. The sustainable upkeep of this Tools and Data Services Registry is assured by a curation effort driven by and tailored to local needs, and shared amongst a network of engaged partners. As of November 2015, the registry includes 1785 resources, with depositions from 126 individual registrations including 52 institutional providers and 74 individuals. With community support, the registry can become a standard for dissemination of information about bioinformatics resources: we welcome everyone to join us in this common endeavour. The registry is freely available at https://bio.tools.

Hydrodynamic chromatography coupled to single-particle ICP-MS for the simultaneous characterization of AgNPs and determination of dissolved Ag in plasma and blood of burn patients

AbstractSilver nanoparticles (AgNPs) are increasingly used in medical devices as innovative antibacterial agents, but no data are currently available on their chemical transformations and fate in vivo in the human body, particularly on their potential to reach the circulatory system. To study the processes involving AgNPs in human plasma and blood, we developed an analytical method based on hydrodynamic chromatography (HDC) coupled to inductively coupled plasma mass spectrometry (ICP-MS) in single-particle detection mode. An innovative algorithm was implemented to deconvolute the signals of dissolved Ag and AgNPs and to extrapolate a multiparametric characterization of the particles in the same chromatogram. From a single injection, the method provides the concentration of dissolved Ag and the distribution of AgNPs in terms of hydrodynamic diameter, mass-derived diameter, number and mass concentration. This analytical approach is robust and suitable to study quantitatively the dynamics and kinetics of AgNPs in complex biological fluids, including processes such as agglomeration, dissolution and formation of protein coronas. The method was applied to study the transformations of AgNP standards and an AgNP-coated dressing in human plasma, supported by micro X-ray fluorescence (μXRF) and micro X-ray absorption near-edge spectroscopy (μXANES) speciation analysis and imaging, and to investigate, for the first time, the possible presence of AgNPs in the blood of three burn patients treated with the same dressing. Together with our previous studies, the results strongly support the hypothesis that the systemic mobilization of the metal after topical administration of AgNPs is driven by their dissolution in situ. Graphical AbstractSimplified scheme of the combined analytical approach adopted for studying the chemical dynamics of AgNPs in human plasma/blood

Looking for putative phenoloxidases of compound ascidians: Haemocyanin-like proteins in Polyandrocarpa misakiensis and Botryllus schlosseri

Phenoloxidases (POs) and haemocyanins constitute a family of copper-containing proteins widely distributed among invertebrates. Both of them are able, under appropriate conditions, to convert polyphenols to quinones and induce cytotoxicity through the production of reactive oxygen species, a fundamental event in many immune responses. In ascidians, PO activity has been described and studied in both solitary and colonial species and the enzyme is involved in inflammatory and cytotoxic reactions against foreign cells or molecules, and in the formation of the cytotoxic foci which characterise the nonfusion reaction of botryllids. Expressed genes for two putative POs (CiPO1 and CiPO2) have been recently identified in C. intestinalis. In the present study, we determined the cDNA sequences of two haemocyanin-like proteins from two colonial ascidians: Botryllus schlosseri from the Mediterranean Sea and Polyandrocarpa misakiensis from Japan. Multiple sequence alignments evidenced the similarity between the above sequences and crustacean proPOs whereas the analysis of the three-dimensional structure reveals high similarity with arthropod haemocyanins which share common precursors with arthropod proPOs. Botryllus HLP grouped in the same cluster with Ciona POs, whereas Polyandrocarpa HLP clustered with arthropod haemocyanins; all of them share the full conservation of the six histidines at the two copper-binding sites as well as of other motifs, also found in arthropod haemocyanin subunits, involved in the regulation of enzyme activity. In situ hybridisation indicated that the genes are transcribed inside morula cells, a characteristic haemocyte type in ascidians where PO activity is located, at the beginning of their differentiation. These results represent a first attempt to identify candidate molecules responsible of the PO activity in compound ascidians.

Size and metal composition characterization of nano- and microparticles in tattoo inks by a combination of analytical techniques

We have applied different analytical techniques for studying the size and metal composition of nanoparticles (NPs) and microparticles in tattoo inks. The morphology and size distribution of particles were determined by transmission electron microscopy (TEM) and dynamic light scattering (DLS). These techniques allowed us to obtain size information on all particles in tattoo mixtures. Higher resolution characterization of the size distributions was achieved by means of asymmetric flow field fractionation (AF4) combined with multi-angle light scattering (MALS) detection, which allowed for particle fraction separation and sizing. The high sensitivity and multi-elemental capability of inductively coupled plasma mass spectrometry (ICP-MS) enabled the off-line determination of the metal concentration of the particles in the AF4 fractions. Counting and sizing of particles were also performed by single particle (SP)-ICP-MS. The TEM images of the tattoo inks indicated the presence of individual particles and of aggregates and agglomerates of different shapes; carbon black was recognized in black inks and TiO2 in lighter shades. The DLS measurements showed that the inks were polydispersed with particles continuously distributed from tens to hundreds of nanometers (the Z-average hydrodynamic diameter range was 31–421 nm). The use of AF4 revealed the presence of two different fractions of particles in colored inks and one fraction in black inks, with mean sizes ranging from ca. 50 nm to ca. 400 nm. AF4 and off-line ICP-MS analyses of these fractions showed micronic and sub-micronic Al and Ti particles and Cu at the nanometer level (<100 nm). SP-ICP-MS analysis confirmed these latter findings: the inks contained nanosized materials of Cu (such as CuO or Cu-phthalocyanine), while Al (as Al2O3) and Ti (as TiO2) were detected as larger sized particles. Overall, this work confirms the power of different analytical techniques in detecting and characterizing NPs and microparticles in tattoo inks. We show that tattoo ink suspensions unquestionably contain NPs, suggesting the need for an assessment of how they interact with human skin as well as if they can penetrate the derma to reach the bloodstream.

Quaternary Structure Heterogeneity of Oligomeric Proteins: A SAXS and SANS Study of the Dissociation Products of Octopus vulgaris Hemocyanin

Octopus vulgaris hemocyanin shows a particular self-assembling pattern, characterized by a hierarchical organization of monomers. The highest molecular weight aggregate is a decamer, the stability of which in solution depends on several parameters. Different pH values, buffer compositions, H2O/D2O ratios and Hofmeister’s salts result in modifications of the aggregation state of Octopus vulgaris hemocyanin. The new QUAFIT method, recently applied to derive the structure of the decameric and the monomeric assembly from small-angle scattering data, is used here to model the polydisperse system that results from changing the solution conditions. A dataset of small-angle X-rays and neutron scattering curves is analysed by QUAFIT to derive structure, composition and concentration of different assemblies present in solution. According to the hierarchy of the association/dissociation processes and the possible number of different aggregation products in solution, each sample has been considered as a heterogeneous mixture composed of the entire decamer, the dissociated “loose” monomer and all the intermediate dissociation products. Scattering curves corresponding to given experimental conditions are well fitted by using a linear combination of single particle form factors. QUAFIT has proved to be a method of general validity to describe solutions of proteins that, even after purification processes, result to be intrinsically heterogeneous.

Solution structures of 2 6-meric and 4 6-meric hemocyanins of crustaceans Carcinus aestuarii, Squilla mantis and Upogebia pusilla

Arthropod hemocyanins (Hcs) are a family of large, high molecular mass, extracellular oxygen transport proteins. They form oligomeric quaternary structures based on different arrangements of a basic 6×75 kDa hexameric unit. Their complex quaternary structures present binding sites for allosteric effectors and regulate the oxygen binding process in a cooperative manner. In order to describe the functional regulation of arthropod Hcs, a detailed description of their quaternary structure is necessary. We have utilized small angle X-ray scattering to characterize the structure of three arthropod Hcs in unperturbed conditions. Two different levels of complexity are evaluated: for the 2×6-meric case, we analyzed the Hcs of the portunid crab Carcinus aestuarii and stomatopod Squilla mantis, while in the case of 4×6-meric structures, we studied the Hc of the thalassinid shrimp Upogebia pusilla. While C. aestuarii Hc presented a structure comparable to other 2×6-meric crustacean Hcs, S. mantis Hc shows a peculiar and quite unique arrangement of its building blocks, resembling a substructure of giant Hcs found among cheliceratans. For U. pusilla, the arrangement of its subunits is described as tetrahedral, in contrast to the more common square planar 4×6-meric structure found in other arthropod Hcs.

MobiDB 3.0: More annotations for intrinsic disorder, conformational diversity and interactions in proteins

Abstract The MobiDB (URL: mobidb.bio.unipd.it) database of protein disorder and mobility annotations has been significantly updated and upgraded since its last major renewal in 2014. Several curated datasets for intrinsic disorder and folding upon binding have been integrated from specialized databases. The indirect evidence has also been expanded to better capture information available in the PDB, such as high temperature residues in X-ray structures and overall conformational diversity. Novel nuclear magnetic resonance chemical shift data provides an additional experimental information layer on conformational dynamics. Predictions have been expanded to provide new types of annotation on backbone rigidity, secondary structure preference and disordered binding regions. MobiDB 3.0 contains information for the complete UniProt protein set and synchronization has been improved by covering all UniParc sequences. An advanced search function allows the creation of a wide array of custom-made datasets for download and further analysis. A large amount of information and cross-links to more specialized databases are intended to make MobiDB the central resource for the scientific community working on protein intrinsic disorder and mobility.