Enhancement of DNA uptake in FUT8‐deleted CHO cells for transient production of afucosylated antibodies

Removal of the core α1,6 fucose from the glycans in the Fc region of IgG1 antibodies has been demonstrated to improve antibody‐dependent cellular cytotoxicity (ADCC) activity. In order to produce afucosylated antibodies using transient transfection, a FUT8 knockout (FUT8KO) cell line was generated in a CHO host cell line using the zinc finger nuclease technology. Transient transfection of DNA into mammalian cells using the cationic polymer, polyethylenimine (PEI), is commonly used for rapid generation of recombinant proteins. FUT8KO cells evaluated in PEI transfections yielded lower titers than parental CHO WT cells. FACS and HPLC analyses revealed that the FUT8KO cells had lower cell surface heparan sulfate (HS) levels than CHO WT. Removal of cell surface HS resulted in reduced uptake of PEI‐transfected DNA (PEI:DNA) and lower transfection titers suggesting that PEI:DNA relies on HS for binding and cellular entry. The absence of cell surface HS did not severely impact transfections performed with cationic liposomes. We undertook two approaches to improve transient production of afucosylated antibodies. First, we evaluated transfection of FUT8KO cells with cationic liposomes, which were observed to be less dependent on HS levels for uptake. Transfection of FUT8KO cells using the cationic liposome, DMRIE‐C, produced similar titers to CHO WT in both shake flask and large‐scale 10 L bioreactors. The second approach was to engineer a cell line overexpressing exostosin‐1 (EXT1), an enzyme responsible for HS chain elongation, to increase HS content. EXT1‐FUT8KO and CHO WT cells produced comparable levels of antibody from PEI transfections. Biotechnol. Bioeng. 2010;106: 751–763. © 2010 Wiley Periodicals, Inc.     

The trypanosome Pumilio-domain protein PUF7 associates with a nuclear cyclophilin and is involved in ribosomal RNA maturation

Proteins with Pumilio RNA binding domains (Puf proteins) are ubiquitous in eukaryotes. Some Puf proteins bind to the 3′-untranslated regions of mRNAs, acting to repress translation and promote degradation; others are involved in ribosomal RNA maturation. The genome of Trypanosoma brucei encodes eleven Puf proteins whose function cannot be predicted by sequence analysis. We show here that epitope-tagged TbPUF7 is located in the nucleolus, and associated with a nuclear cyclophilin-like protein, TbNCP1. RNAi targeting PUF7 reduced trypanosome growth and inhibited two steps in ribosomal RNA processing.     

Enzymatic activation of sulfur for incorporation into biomolecules in prokaryotes

Sulfur is a functionally important element of living matter. Incorporation into biomolecules occurs by two basic strategies. Sulfide is added to an activated acceptor in the biosynthesis of cysteine, from which methionine, coenzyme A and a number of biologically important thiols can be constructed. By contrast, the biosyntheses of iron sulfur clusters, cofactors such as thiamin, molybdopterin, biotin and lipoic acid, and the thio modification of tRNA require an activated sulfur species termed persulfidic sulfur (R-S-SH) instead of sulfide. Persulfidic sulfur is produced enzymatically with the IscS protein, the SufS protein and rhodanese being the most prominent biocatalysts. This review gives an overview of sulfur incorporation into biomolecules in prokaryotes with a special emphasis on the properties and the enzymatic generation of persulfidic sulfur as well as its use in biosynthetic pathways.     

High-affinity AKAP7delta-protein kinase A interaction yields novel protein kinase A-anchoring disruptor peptides

PKA (protein kinase A) is tethered to subcellular compartments by direct interaction of its regulatory subunits (RI or RII) with AKAPs (A kinase-anchoring proteins). AKAPs preferentially bind RII subunits via their RII-binding domains. RII-binding domains form structurally conserved amphipathic helices with unrelated sequences. Their binding affinities for RII subunits differ greatly within the AKAP family. Amongst the AKAPs that bind RIIalpha subunits with high affinity is AKAP7delta [AKAP18delta; K(d) (equilibrium dissociation constant) value of 31 nM]. An N-terminally truncated AKAP7delta mutant binds RIIalpha subunits with higher affinity than the full-length protein presumably due to loss of an inhibitory region [Henn, Edemir, Stefan, Wiesner, Lorenz, Theilig, Schmidtt, Vossebein, Tamma, Beyermann et al. (2004) J. Biol. Chem. 279, 26654-26665]. In the present study, we demonstrate that peptides (25 amino acid residues) derived from the RII-binding domain of AKAP7delta bind RIIalpha subunits with higher affinity (K(d)=0.4+/-0.3 nM) than either full-length or N-terminally truncated AKAP7delta, or peptides derived from other RII binding domains. The AKAP7delta-derived peptides and stearate-coupled membrane-permeable mutants effectively disrupt AKAP-RII subunit interactions in vitro and in cell-based assays. Thus they are valuable novel tools for studying anchored PKA signalling. Molecular modelling indicated that the high affinity binding of the amphipathic helix, which forms the RII-binding domain of AKAP7delta, with RII subunits involves both the hydrophobic and the hydrophilic faces of the helix. Alanine scanning (25 amino acid peptides, SPOT technology, combined with RII overlay assays) of the RII binding domain revealed that hydrophobic amino acid residues form the backbone of the interaction and that hydrogen bond- and salt-bridge-forming amino acid residues increase the affinity of the interaction.     

Emerging Shiga Toxin-Producing Escherichia coli Serotypes in Europe: O100:H- and O127:H40

Novel and as yet rare non-O157 Shiga toxin (Stx)-producing Escherichia coli (STEC) serotypes are emerging in Europe. Two different sorbitol-fermenting STECs, O100:H- carrying the virulence gene stx2 and O127:H40 carrying stx1 and eae genes (found in two related subjects), were isolated from patients’ stool samples. Non-O157 STEC infections in humans are currently under-diagnosed. This report highlights the need for, and importance of, screening for Shiga toxins or serotypes other than just O157.     

Radioiodinated clioquinol as a biomarker for beta-amyloid: Zn complexes in Alzheimer's disease

Neocortical beta-amyloid (Abeta) aggregates in Alzheimer's disease (AD) are enriched in transition metals that mediate assembly. Clioquinol (CQ) targets metal interaction with Abeta and inhibits amyloid pathology in transgenic mice. Here, we investigated the binding properties of radioiodinated CQ ([(125)I]CQ) to different in vitro and in vivo Alzheimer models. We observed saturable binding of [(125)I]CQ to synthetic Abeta precipitated by Zn(2+) (K(d)=0.45 and 1.40 nm for Abeta(1-42) and Abeta(1-40), respectively), which was fully displaced by free Zn(2+), Cu(2+), the chelator DTPA (diethylene triamine pentaacetic acid) and partially by Congo red. Sucrose density gradient of post-mortem AD brain indicated that [(125)I]CQ concentrated in a fraction enriched for both Abeta and Zn, which was modulated by exogenous addition of Zn(2+) or DTPA. APP transgenic (Tg2576) mice injected with [(125)I]CQ exhibited higher brain retention of tracer compared to non-Tg mice. Autoradiography of brain sections of these animals confirmed selective [(125)I]CQ enrichment in the neocortex. Histologically, both thioflavine-S (ThS)-positive and negative structures were labeled by [(125)I]CQ. A pilot SPECT study of [(123)I]CQ showed limited uptake of the tracer into the brain, which did however, appear to be more rapid in AD patients compared to age-matched controls. These data support metallated Abeta species as the neuropharmacological target of CQ and indicate that this drug class may have potential as in vivo imaging agents for Alzheimer neuropathology.     

The CDK5 repressor CDK5RAP1 is a methylthiotransferase acting on nuclear and mitochondrial RNA

The unusual cyclin-dependent protein kinase 5 (CDK5) was discovered based on its sequence homology to cell cycle regulating CDKs. CDK5 was found to be active in brain tissues, where it is not involved in cell cycle regulation but in the regulation of neuronal cell differentiation and neurocytoskeleton dynamics. An aberrant regulation of CDK5 leads to the development of various neurodegenerative diseases including Alzheimer's disease. Although CDK5 is not regulated by cyclins, its activity does depend on the association with a protein activator and the presence or absence of further inhibitory factors. Recently, CDK5RAP1 was discovered to inhibit the active CDK5 kinase. Here, we show that CDK5RAP1 is a radical SAM enzyme, which postsynthetically converts the RNA modification N6-isopentenyladenosine (i(6)A) into 2-methylthio-N6-isopentenyladenosine (ms(2)i(6)A). This conversion is surprisingly not limited to mitochondrial tRNA, where the modification was known to exist. Instead, CDK5RAP1 introduces the modification also into nuclear RNA species establishing a link between postsynthetic kinase-based protein modification and postsynthetic RNA modification.     

9-Methyl-β-carboline-induced cognitive enhancement is associated with elevated hippocampal dopamine levels and dendritic and synaptic proliferation

β-Carbolines (BCs) belong to the heterogenous family of carbolines, which have been found exogenously, that is, in various fruits, meats, tobacco smoke, alcohol and coffee, but also endogenously, that is, blood, brain and CSF. These exogenous and endogenous BCs and some of their metabolites can exert neurotoxic effects, however, an unexpected stimulatory effect of 9-methyl-β-carboline (9-me-BC) on dopaminergic neurons in primary mesencephalic cultures was recently discovered. The aim of the present study was to extend our knowledge on the stimulatory effects of 9-me-BC and to test the hypothesis that 9-me-BC may act as a cognitive enhancer. We found that 10 days (but not 5 days) of pharmacological treatment with 9-me-BC (i) improves spatial learning in the radial maze, (ii) elevates dopamine levels in the hippocampal formation, and (iii) results after 10 days of treatment in elongated, more complex dendritic trees and higher spine numbers on granule neurons in the dentate gyrus of 9-me-BC-treated rats. Our results demonstrate that beyond its neuroprotective/neurorestorative and anti-inflammatory effects, 9-me-BC acts as a cognitive enhancer in a hippocampus-dependent task, and that the behavioral effects may be associated with a stimulatory impact on hippocampal dopamine levels and dendritic and synaptic proliferation.