Jörg Kobarg

Size-selective silver nanoparticles: future of biomedical devices with enhanced bactericidal properties

Silver nanoparticles (AgNPs) are attracting attention due to their bactericidal activity and consequent possible biomedical applications. The key to their broad-acting and potent biocidal property seems to be based on the size-related mechanism by which AgNPs act on different bacteria strains. Here, we report the synthesis and successful size-selective fractionation of AgNPs obtained through chemical reduction of silver nitrate in ethylene glycol using polyvinylpyrrolidone as a protective agent. A combination of characterization techniques (UV-vis spectroscopy, transmission electron microscopy and small-angle X-ray scattering) is employed to differentiate the two size-fractionated samples. From the analyses, it is evidenced that AgNPs are mainly spherical and have their radius centered at ∼8.5 and ∼11.0 nm. The nanoparticles bactericidal efficacy is investigated using the disk diffusion test against Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis and Micrococcus lysodeikticus. Although both fractionated samples present bactericidal activity against all four tested bacteria (one Gram negative and three Gram positives), those presenting smaller size own enhanced antibacterial properties.

A draft of the human septin interactome.

Background Septins belong to the GTPase superclass of proteins and have been functionally implicated in cytokinesis and the maintenance of cellular morphology. They are found in all eukaryotes, except in plants. In mammals, 14 septins have been described that can be divided into four groups. It has been shown that mammalian septins can engage in homo- and heterooligomeric assemblies, in the form of filaments, which have as a basic unit a hetero-trimeric core. In addition, it has been speculated that the septin filaments may serve as scaffolds for the recruitment of additional proteins. Methodology/Principal Findings Here, we performed yeast two-hybrid screens with human septins 1–10, which include representatives of all four septin groups. Among the interactors detected, we found predominantly other septins, confirming the tendency of septins to engage in the formation of homo- and heteropolymeric filaments. Conclusions/Significance If we take as reference the reported arrangement of the septins 2, 6 and 7 within the heterofilament, (7-6-2-2-6-7), we note that the majority of the observed interactions respect the “group rule”, i.e. members of the same group (e.g. 6, 8, 10 and 11) can replace each other in the specific position along the heterofilament. Septins of the SEPT6 group preferentially interacted with septins of the SEPT2 group (p<0.001), SEPT3 group (p<0.001) and SEPT7 group (p<0.001). SEPT2 type septins preferentially interacted with septins of the SEPT6 group (p<0.001) aside from being the only septin group which interacted with members of its own group. Finally, septins of the SEPT3 group interacted preferentially with septins of the SEPT7 group (p<0.001). Furthermore, we found non-septin interactors which can be functionally attributed to a variety of different cellular activities, including: ubiquitin/sumoylation cycles, microtubular transport and motor activities, cell division and the cell cycle, cell motility, protein phosphorylation/signaling, endocytosis, and apoptosis.

Characterization of a new family of proteins that interact with the C‐terminal region of the chromatin‐remodeling factor CHD‐31

The two human proteins Ki‐1/57 and CGI‐55 have highly similar amino acid sequences but their functions are unknown. We analyzed them by yeast two‐hybrid screens and found that they interact with the C‐terminal region of the human chromatin‐remodeling factor CHD‐3 (chromo‐helicase‐DNA‐binding domain protein‐3). The interaction of CGI‐55 and CHD‐3 could be confirmed in vitro and in vivo by co‐immunoprecipitations from Sf9 insect cells. Mapping showed that CGI‐55 interacts with CHD‐3 via two regions at its N‐ and C‐terminals. The CGI‐55 and Ki‐1/57 mRNAs show highest expression in muscle, colon and kidney. A CGI55–GFP fusion protein was localized in the cytoplasm, nucleus and perinuclear regions of HeLa cells. These data suggest the possibility that CGI‐55 and Ki‐1/57 might be involved in nuclear functions like the remodeling of chromatin.

FEZ1 Dimerization and Interaction with Transcription Regulatory Proteins Involves Its Coiled-coil Region

The fasciculation and elongation protein ζ1 (FEZ1) is a mammalian orthologue of the Caenorhabditis elegans protein UNC-76, which is necessary for axon growth in that nematode. In previous studies FEZ1 has been found to interact with protein kinase Cζ, DISC1, the agnoprotein of the human polyomavirus JC virus, and E4B, a U-box-type ubiquitin-protein isopeptide ligase. We reported previously that FEZ1 and its paralogue FEZ2 are proteins that interact with NEK1, a protein kinase involved in polycystic kidney disease and DNA repair mechanisms at the G2/M phase of the cell cycle. Here we report the identification of 16 proteins that interact with human FEZ1-(221–396) in a yeast two-hybrid assay of a human fetal brain cDNA library. The 13 interacting proteins of known functions take part either in transcription regulation and chromatin remodeling (6 proteins), the regulation of neuronal cell development (2 proteins) and cellular transport mechanisms (3 proteins) or participate in apoptosis (2 proteins). We were able to confirm eight of the observed interactions by in vitro pull-down assays with recombinant fusion proteins. The confirmed interacting proteins include FEZ1 itself and three transcription controlling proteins (SAP30L, DRAP1, and BAF60a). In mapping studies we found that the C-terminal regions of FEZ1, and especially its coiled-coil region, are involved in its dimerization, its heterodimerization with FEZ2, and in the interaction with 10 of the identified interacting proteins. Our results give further support to the previous speculation of the functional involvement of FEZ1 in neuronal development but suggest further that FEZ1 may also be involved in transcriptional control.

Tailored Silica-Antibiotic Nanoparticles: Overcoming Bacterial Resistance with Low Cytotoxicity

New and more aggressive antibiotic resistant bacteria arise at an alarming rate and represent an ever-growing challenge to global health care systems. Consequently, the development of new antimicrobial agents is required to overcome the inefficiency of conventional antibiotics and bypass treatment limitations related to these pathologies. In this study, we present a synthesis protocol, which was able to entrap tetracycline antibiotic into silica nanospheres. Bactericidal efficacy of these structures was tested against bacteria that were susceptible and resistant to antibiotics. For nonresistant bacteria, our composite had bactericidal efficiency comparable to that of free-tetracycline. On the other hand, the synthesized composites were able to avoid bacterial growth of resistant bacteria while free-tetracycline has shown no significant bactericidal effect. Finally, we have investigated the cytotoxicity of these nanoparticles against mammalian cells to check any possible poisoning effect. It was found that these nanospheres are not apoptosis-inducers and only a reduction on the cell replication rate was seen when compared to the control without nanoparticles.

CGI-55 interacts with nuclear proteins and co-localizes to p80-coilin positive-coiled bodies in the nucleus

The human protein CGI-55 has been described as a chromo-helicase-DNA-binding domain protein (CHD)-3 interacting protein and was also found to interact with the 3′-region of the plasminogen activator inhibitor (PAI)-1 mRNA. Here, we used CGI-55 as a “bait” in a yeast two-hybrid screen and identified eight interacting proteins: Dax, Topoisomerase I binding RS (Topors), HPC2, UBA2, TDG, and protein inhibitor of activated STAT (signal transducer and activator of transcription) (PIAS)-1,-3, and-y. These proteins are either structurally or functionally associated with promyelocytic leukemia nuclear bodies (PML-NBs), protein sumoylation, or the regulation of transcription. The interactions of CGI-55 with Daxx, Topors, PIASy, and UBA2 were confirmed by in vivo colocalization experiments in HeLa cells, by using green (GFP) and red fluorescence fusion proteins. A mapping study of the CGI-55 binding site for these proteins revealed three distinct patterns of interaction. The fact that CGI-55-GFP has been localized in cytoplasm and nucleus in a dotted manner, and its interaction with proteins associated with PML-NBs, suggested that CGI-55 might be associated with nuclear bodies. Although Daxx and Topors co-localized with promyelocytic leukemia protein (PML), CGI-55 itself as well as PIASy and UBA2 showed only little co-localization with PML. However, we observed that CGI-55 localizes to the nucleolus and co-localizes with p80-coilin positive nuclear-coiled bodies.

Potential Antileukemia Effect and Structural Analyses of SRPK Inhibition by N-(2-(Piperidin-1-yl)-5-(Trifluoromethyl)Phenyl)Isonicotinamide (SRPIN340).

Dysregulation of pre-mRNA splicing machinery activity has been related to the biogenesis of several diseases. The serine/arginine-rich protein kinase family (SRPKs) plays a critical role in regulating pre-mRNA splicing events through the extensive phosphorylation of splicing factors from the family of serine/arginine-rich proteins (SR proteins). Previous investigations have described the overexpression of SRPK1 and SRPK2 in leukemia and other cancer types, suggesting that they would be useful targets for developing novel antitumor strategies. Herein, we evaluated the effect of selective pharmacological SRPK inhibition by N-(2-(piperidin-1-yl)-5-(trifluoromethyl)phenyl)isonicotinamide (SRPIN340) on the viability of lymphoid and myeloid leukemia cell lines. Along with significant cytotoxic activity, the effect of treatments in regulating the phosphorylation of the SR protein family and in altering the expression of MAP2K1, MAP2K2, VEGF and FAS genes were also assessed. Furthermore, we found that pharmacological inhibition of SRPKs can trigger early and late events of apoptosis. Finally, intrinsic tryptophan fluorescence emission, molecular docking and molecular dynamics were analyzed to gain structural information on the SRPK/SRPIN340 complex. These data suggest that SRPK pharmacological inhibition should be considered as an alternative therapeutic strategy for fighting leukemias. Moreover, the obtained SRPK-ligand interaction data provide useful structural information to guide further medicinal chemistry efforts towards the development of novel drug candidates.

Ki-1/57 interacts with PRMT1 and is a substrate for arginine methylation

The human 57 kDa Ki‐1 antigen (Ki‐1/57) is a cytoplasmic and nuclear protein, associated with Ser/Thr protein kinase activity, and phosphorylated at the serine and threonine residues upon cellular activation. We have shown that Ki‐1/57 interacts with chromo‐helicase DNA‐binding domain protein 3 and with the adaptor/signaling protein receptor of activated kinase 1 in the nucleus. Among the identified proteins that interacted with Ki‐1/57 in a yeast two‐hybrid system was the protein arginine‐methyltransferase‐1 (PRMT1). Most interestingly, when PRMT1 was used as bait in a yeast two‐hybrid system we were able to identify Ki‐1/57 as prey among 14 other interacting proteins, the majority of which are involved in RNA metabolism or in the regulation of transcription. We found that Ki‐1/57 and its putative paralog CGI‐55 have two conserved Gly/Arg‐rich motif clusters (RGG/RXR box, where X is any amino acid) that may be substrates for arginine‐methylation by PRMT1. We observed that all Ki‐1/57 protein fragments containing RGG/RXR box clusters interact with PRMT1 and are targets for methylation in vitro. Furthermore, we found that Ki‐1/57 is a target for methylation in vivo. Using immunofluorescence experiments we observed that treatment of HeLa cells with an inhibitor of methylation, adenosine‐2′,3′‐dialdehyde (Adox), led to a reduction in the cytoplasmic immunostaining of Ki‐1/57, whereas its paralog CGI‐55 was partially redistributed from the nucleus to the cytoplasm upon Adox treatment. In summary, our data show that the yeast two‐hybrid assay is an effective system for identifying novel PRMT arginine‐methylation substrates and may be successfully applied to other members of the growing family of PRMTs.

Expression of deletion mutants of the hepatitis B virus protein HBx in E. coli and characterization of their RNA binding activities.

The hepatitis B virus protein HBx has been implicated in the development of liver cancer. It has been shown that the HBx protein is able to bind to single-stranded DNA in a specific manner. This DNA binding activity might be relevant for HBx oncogene character. To study the HBx interaction with nucleic acids in more detail we expressed full-length HBx as well as several N- and C-terminally truncated HBx proteins as 6xHis and GST-fusions in E. coli. Using a gel shift assay, we were able to demonstrate that all of the truncated HBx proteins have the ability to bind to an AU-rich RNA. The affinity of GST-HBx #3 (residues 80-142) was an order of magnitude higher than that of GST-HBx #2 (residues 5-79), indicating that a high affinity RNA binding site is located in HBx C-terminal half. AUF1 is the protein ligand that binds to AU-rich RNA regions present in certain proto-oncogene mRNAs and causes their rapid degradation. By a competitive binding experiment of AUF1 and HBx to the AU-rich RNA oligonucleotide, we show that HBx is able to displace AUF1 from its binding site on the RNA oligonucleotide. This new aspect of HBx function is discussed in the context of cellular transformation.

Characterization of the Human NEK7 Interactome Suggests Catalytic and Regulatory Properties Distinct from Those of NEK6

Human NEK7 is a regulator of cell division and plays an important role in growth and survival of mammalian cells. Human NEK6 and NEK7 are closely related, consisting of a conserved C-terminal catalytic domain and a nonconserved and disordered N-terminal regulatory domain, crucial to mediate the interactions with their respective proteins. Here, in order to better understand NEK7 cellular functions, we characterize the NEK7 interactome by two screening approaches: one using a yeast two-hybrid system and the other based on immunoprecipitation followed by mass spectrometry analysis. These approaches led to the identification of 61 NEK7 interactors that contribute to a variety of biological processes, including cell division. Combining additional interaction and phosphorylation assays from yeast two-hybrid screens, we validated CC2D1A, TUBB2B, MNAT1, and NEK9 proteins as potential NEK7 interactors and substrates. Notably, endogenous RGS2, TUBB, MNAT1, NEK9, and PLEKHA8 localized with NEK7 at key sites throughout the cell cycle, especially during mitosis and cytokinesis. Furthermore, we obtained evidence that the closely related kinases NEK6 and NEK7 do not share common interactors, with the exception of NEK9, and display different modes of protein interaction, depending on their N- and C-terminal regions, in distinct fashions. In summary, our work shows for the first time a comprehensive NEK7 interactome that, combined with functional in vitro and in vivo assays, suggests that NEK7 is a multifunctional kinase acting in different cellular processes in concert with cell division signaling and independently of NEK6.