Inorganic nanoparticles for transfection of mammalian cells and removal of viruses from aqueous solutions

Owing to their small size, synthetic nanoparticles show unprecedented biophysical and biochemical properties which may foster novel advances in life-science research. Using flame-spray synthesis technology we have produced non-coated aluminum-, calcium-, cerium-, and zirconium-derived inorganic metal oxide nanoparticles which not only exhibit high affinity for nucleic acids, but can sequester such compounds from aqueous solution. This non-covalent DNA-binding capacity was successfully used to transiently transfect a variety of mammalian cells including human, reaching transfection efficiencies which compared favorably with classic calcium phosphate precipitation (CaP) procedures and lipofection. In this straightforward protocol, transfection was enabled by simply mixing nanoparticles with DNA in solution prior to addition to the target cell population. Transiently transfected cells showed higher production levels of the human secreted glycoprotein SEAP compared to isogenic populations transfected with established technologies. Inorganic metal oxide nanoparticles also showed a high binding capacity to human-pathogenic viruses including adenovirus, adeno-associated virus and human immunodeficiency virus type 1 and were able to clear these pathogens from aqueous solutions. The DNA transfection and viral clearance capacities of inorganic metal oxide nanoparticles may provide cost-effective biopharmaceutical manufacturing and water treatment in developing countries.     

Biophysics and bioinformatics reveal structural differences of the two peripheral stalk subunits in chloroplast ATP synthase

ATP synthases convert an electrochemical proton gradient into rotational movement to produce the ubiquitous energy currency adenosine triphosphate. Tension generated by the rotational torque is compensated by the stator. For this task, a peripheral stalk flexibly fixes the hydrophilic catalytic part F1 to the membrane integral proton conducting part F(O) of the ATP synthase. While in eubacteria a homodimer of b subunits forms the peripheral stalk, plant chloroplasts and cyanobacteria possess a heterodimer of subunits I and II. To better understand the functional and structural consequences of this unique feature of photosynthetic ATP synthases, a procedure was developed to purify subunit I from spinach chloroplasts. The secondary structure of subunit I, which is not homologous to bacterial b subunits, was compared to heterologously expressed subunit II using CD and FTIR spectroscopy. The content of alpha-helix was determined by CD spectroscopy to 67% for subunit I and 41% for subunit II. In addition, bioinformatics was applied to predict the secondary structure of the two subunits and the location of the putative coiled-coil dimerization regions. Three helical domains were predicted for subunit I and only two uninterrupted domains for the shorter subunit II. The predicted length of coiled-coil regions varied between different species and between subunits I and II.     

Labeling, detection and identification of newly synthesized proteomes with bioorthogonal non-canonical amino-acid tagging

A major aim of proteomics is the identification of proteins in a given proteome at a given metabolic state. This protocol describes the step-by-step labeling, purification and detection of newly synthesized proteins in mammalian cells using the non-canonical amino acid azidohomoalanine (AHA). In this method, metabolic labeling of newly synthesized proteins with AHA endows them with the unique chemical functionality of the azide group. In the subsequent click chemistry tagging reaction, azide-labeled proteins are covalently coupled to an alkyne-bearing affinity tag. After avidin-based affinity purification and on-resin trypsinization, the resulting peptide mixture is subjected to tandem mass spectrometry for identification. In combination with deuterated leucine-based metabolic colabeling, candidate proteins can be immediately validated. Bioorthogonal non-canonical amino-acid tagging can be combined with any subcellular fractionation, immunopurification or other proteomic method to identify specific subproteomes, thereby reducing sample complexity and enabling the identification of subtle changes in a proteome. This protocol can be completed in 5 days.     

The myotonic dystrophy type 2 protein ZNF9 is part of an ITAF complex that promotes cap-independent translation

The 5'-untranslated region of the ornithine decarboxylase (ODC) mRNA contains an internal ribosomal entry site (IRES). Mutational analysis of the ODC IRES has led to the identification of sequences necessary for cap-independent translation of the ODC mRNA. To discover novel IRES trans-acting factors (ITAFs), we performed a proteomics screen for proteins that regulate ODC translation using the wild-type ODC mRNA and a mutant version with an inactive IRES. We identified two RNA-binding proteins that associate with the wild-type ODC IRES but not the mutant IRES. One of these RNA-binding proteins, PCBP2, is an established activator of viral and cellular IRESs. The second protein, ZNF9 (myotonic dystrophy type 2 protein), has not been shown previously to bind IRES-like elements. Using a series of biochemical assays, we validated the interaction of these proteins with ODC mRNA. Interestingly ZNF9 and PCBP2 biochemically associated with each other and appeared to function as part of a larger holo-ITAF ribonucleoprotein complex. Our functional studies showed that PCBP2 and ZNF9 stimulate translation of the ODC IRES. Importantly these results may provide insight into the normal role of ZNF9 and why ZNF9 mutations cause myotonic dystrophy.     

In vivo gene regulation in Salmonella spp. by a salicylate-dependent control circuit

Systems allowing tightly regulated expression of prokaryotic genes in vivo are important for performing functional studies of bacterial genes in host-pathogen interactions and establishing bacteria-based therapies. We integrated a regulatory control circuit activated by acetyl salicylic acid (ASA) in attenuated Salmonella enterica that carries an expression module with a gene of interest under control of the XylS2-dependent Pm promoter. This resulted in 20-150-fold induction ex vivo. The regulatory circuit was also efficiently induced by ASA when the bacteria resided in eukaryotic cells, both in vitro and in vivo. To validate the circuit, we administered Salmonella spp., carrying an expression module encoding the 5-fluorocytosine-converting enzyme cytosine deaminase in the bacterial chromosome or in a plasmid, to mice with tumors. Induction with ASA before 5-fluorocytosine administration resulted in a significant reduction of tumor growth. These results demonstrate the usefulness of the regulatory control circuit to selectively switch on gene expression during bacterial infection.     

Meiotic chromosomes in motion: a perspective from Mus musculus and Caenorhabditis elegans

Vigorous chromosome movement during the extended prophase of the first meiotic division is conserved in most eukaryotes. The movement is crucial for the faithful segregation of homologous chromosomes into daughter cells, and thus for fertility. A prerequisite for meiotic chromosome movement is the stable and functional attachment of telomeres or chromosome ends to the nuclear envelope and their cytoplasmic coupling to the cytoskeletal forces responsible for generating movement. Important advances in understanding the components, mechanisms, and regulation of chromosome end attachment and movement have recently been made. This review focuses on insights gained from experiments into two major metazoan model organisms: the mouse, Mus musculus, and the nematode, Caenorhabditis elegans.

     

Formation of the single-layer beta-sheet of Borrelia burgdorferi OspA in the absence of the C-terminal capping globular domain

Borrelia outer surface protein A (OspA) contains a unique single-layer beta-sheet that connects N and C-terminal globular domains. This single-layer beta-sheet segment (beta-strands 8-10) is highly stable in solution, although it is exposed to the solvent on both faces of the sheet and thus it does not contain a hydrophobic core. Here, we tested whether interactions with the C-terminal domain are essential for the formation of the single-layer beta-sheet. We characterized the solution structure, dynamics and stability of an OspA fragment corresponding to beta-strands 1-12 (termed OspA[27-163]), which lacks a majority of the C-terminal globular domain. Analyses of NMR chemical shifts and backbone nuclear Overhauser effect (NOE) connectivities showed that OspA[27-163] is folded except the 12th and final beta-strand. (1)H-(15)N heteronuclear NOE measurements and amide H-(2)H exchange revealed that the single-layer beta-sheet in this fragment is more flexible than the corresponding region in full-length OspA. Thermal-denaturation experiments using differential scanning calorimetry and NMR spectroscopy revealed that the N-terminal globular domain in the fragment has a conformational stability similar to that of the same region in the full-length protein, and that the single-layer beta-sheet region also has a modest thermal stability. These results demonstrate that the unique single-layer beta-sheet retains its conformation in the absence of its interactions with the C-terminal domain. This fragment is significantly smaller than the full-length OspA, and thus it is expected to facilitate studies of the folding mechanism of this unusual beta-sheet structure.     

Cloning and characterization of ADAMTS11, an aggrecanase from the ADAMTS family.

Aggrecan is responsible for the mechanical properties of cartilage. One of the earliest changes observed in arthritis is the depletion of cartilage aggrecan due to increased proteolytic cleavage within the interglobular domain. Two major sites of cleavage have been identified in this region at Asn(341)-Phe(342) and Glu(373)-Ala(374). While several matrix metalloproteinases have been shown to cleave at Asn(341)-Phe(342), an as yet unidentified protein termed "aggrecanase" is responsible for cleavage at Glu(373)-Ala(374) and is hypothesized to play a pivotal role in cartilage damage. We have identified and cloned a novel disintegrin metalloproteinase with thrombospondin motifs that possesses aggrecanase activity, ADAMTS11 (aggrecanase-2), which has extensive homology to ADAMTS4 (aggrecanase-1) and the inflammation-associated gene ADAMTS1. ADAMTS11 possesses a number of conserved domains that have been shown to play a role in integrin binding, cell-cell interactions, and extracellular matrix binding. We have expressed recombinant human ADAMTS11 in insect cells and shown that it cleaves aggrecan at the Glu(373)-Ala(374) site, with the cleavage pattern and inhibitor profile being indistinguishable from that observed with native aggrecanase. A comparison of the structure and expression patterns of ADAMTS11, ADAMTS4, and ADAMTS1 is also described. Our findings will facilitate the study of the mechanisms of cartilage degradation and provide targets to search for effective inhibitors of cartilage depletion in arthritic disease.     

Bruton's tyrosine kinase (Btk) associates with protein kinase C mu

Bruton's tyrosine kinase (Btk) is considered an essential signal transducer in B-cells. Mutational defects are associated with a severe immunodeficiency syndrome, X-chromosome linked agammaglobulinemia (XLA). Here we show by coimmunoprecipitation that a member of the protein kinase C (PKC) family, PKCmu, is constitutively associated with Btk. Neither antigen receptor (Ig) crosslinking nor stimulation of B-cells with phorbol ester or H(2)O(2) affected Btk/PKCmu interaction. GST precipitation analysis revealed association of the Btk pleckstrin/Tec homology domain with PKCmu. Transient overexpression of PKCmu deletion mutants as well as expression of selected PKCmu domains in 293T cells revealed that both the kinase domain and the regulatory C1 region are independently capable of binding to the Btk PH-TH domain. These data show the existence of a PKCmu/Btk complex in vivo and identify two PKCmu domains that participate in Btk interaction.