Cytoskeleton-dependent clustering of membrane-bound prion protein on the cell surface.

Prion diseases are a group of neurodegenerative disorders that infect animals and humans with proteinaceous particles called prions. Prions consist of scrapie prion protein (PrPSc), a misfolded version of the cellular prion protein (PrPC). During disease progression, PrPSc replicates by interacting with PrPC and inducing its conversion to PrPSc. Attachment of PrPC to cellular membranes via a glycosylphosphatidylinositol (GPI) anchor is critical for the conversion of PrPC into PrPSc. However, the mechanisms governing PrPC conversion and replication on the membrane remain largely unclear. Here, a site-selectively modified PrP variant equipped with a fluorescent GPI anchor mimic (PrP-GPI) was employed to directly observe PrP at the cellular membrane in neuronal SH-SY5Y cells. PrP-GPI exhibits a cholesterol-dependent membrane accumulation and a cytoskeleton-dependent mobility. More specifically, inhibition of actin polymerization reduced the diffusion of PrP-GPI indicating protein clustering, which resembles the initial step of PrP aggregation and conversion into its pathogenic isoform. An intact actin cytoskeleton might therefore prevent conversion of PrPC into PrPSc and offer new therapeutic angles.

Prion protein-Semisynthetic prion protein (PrP) variants with posttranslational modifications.

Deciphering the pathophysiologic events in prion diseases is challenging, and the role of posttranslational modifications (PTMs) such as glypidation and glycosylation remains elusive due to the lack of homogeneous protein preparations. So far, experimental studies have been limited in directly analyzing the earliest events of the conformational change of cellular prion protein (PrPC ) into scrapie prion protein (PrPSc ) that further propagates PrPC misfolding and aggregation at the cellular membrane, the initial site of prion infection, and PrP misfolding, by a lack of suitably modified PrP variants. PTMs of PrP, especially attachment of the glycosylphosphatidylinositol (GPI) anchor, have been shown to be crucially involved in the PrPSc formation. To this end, semisynthesis offers a unique possibility to understand PrP behavior invitro and invivo as it provides access to defined site-selectively modified PrP variants. This approach relies on the production and chemoselective linkage of peptide segments, amenable to chemical modifications, with recombinantly produced protein segments. In this article, advances in understanding PrP conversion using semisynthesis as a tool to obtain homogeneous posttranslationally modified PrP will be discussed.

Utility of the Phenacyl Protecting Group in Traceless Protein Semisynthesis through Ligation-Desulfurization Chemistry.

Semisynthesis of proteins via expressed protein ligation is a widely applicable method, even more so because of the possibility of ligation at non-cysteine sites using ß-mercapto amino acids that can be converted to the corresponding native amino acids by desulfurization. A drawback of this ligation- desulfurization approach is the removal of any unprotected native cysteine residues within the ligated protein segments. Here, we show that the phenacyl (PAc) moiety can be successfully used to protect cysteines within recombinantly generated protein segments. As such, this group was selectively appended onto cysteine side chains within bacterially expressed polypeptides following intein cleavage, which reveals a rather sensitive thioester at the C-terminus. The PAc group proved to be compatible with native chemical ligation, radical desulfurization, and reverse-phase HPLC conditions, and was smoothly removed at the end. The utility of the PAc protecting group was then demonstrated by the 'traceless' semisynthesis of two proteins containing one or two native cysteines: human small heat shock protein Hsp27 and murine prion protein.

Semisynthetic prion protein (PrP) variants carrying glycan mimics at position 181 and 197 do not form fibrils.

The prion protein (PrP) is an N-glycosylated protein attached to the outer leaflet of eukaryotic cell membranes via a glycosylphosphatidylinositol (GPI) anchor. Different prion strains have distinct glycosylation patterns and the extent of glycosylation of potentially pathogenic misfolded prion protein (PrPSc) has a major impact on several prion-related diseases (transmissible spongiform encephalopathies, TSEs). Based on these findings it is hypothesized that posttranslational modifications (PTMs) of PrP influence conversion of cellular prion protein (PrPC) into PrPSc and, as such, modified PrP variants are critical tools needed to investigate the impact of PTMs on the pathogenesis of TSEs. Here we report a semisynthetic approach to generate PrP variants modified with monodisperse polyethyleneglycol (PEG) units as mimics of N-glycans. Incorporating PEG at glycosylation sites 181 and 197 in PrP induced only small changes to the secondary structure when compared to unmodified, wildtype PrP. More importantly, in vitro aggregation was abrogated for all PEGylated PrP variants under conditions at which wildtype PrP aggregated. Furthermore, the addition of PEGylated PrP as low as 10 mol% to wildtype PrP completely blocked aggregation. A similar effect was observed for synthetic PEGylated PrP segments comprising amino acids 179-231 alone if these were added to wildtype PrP in aggregation assays. This behavior raises the question if large N-glycans interfere with aggregation in vivo and if PEGylated PrP peptides could serve as potential therapeutics.

Semisynthesis of Membrane-Attached Proteins Using Split Inteins.

The site-selective installation of lipid modifications on proteins is critically important in our understanding of how membrane association influences the biophysical properties of proteins as well as to study certain proteins in their native environment. Here, we describe the use of split inteins for the C-terminal attachment of lipid-modified peptides to virtually any protein of interest (POI) via protein trans-splicing (PTS). To achieve this, the protein of interest is expressed in fusion with the N-terminal split intein segment and the C-terminal split intein segment is prepared by solid phase peptide synthesis. A synthetic peptide carrying two lipid chains is also made chemically to serve as a membrane anchor and subsequently linked to the C-terminal split intein by native chemical ligation. Proteins of interest for our work are the prion protein as well as small GTPases; however, extensions to other POIs are possible. Detailed information for the C-terminal introduction of a lipidated membrane anchor (MA) peptide using split intein systems from Synechocystis spp. and Nostoc punctiforme for the Prion protein (PrP, as a challenging protein of interest) and the enhanced green-fluorescent protein (eGFP, as an easily trackable target protein) are provided here.

Biosynthesis of the Fluorinated Natural Product Nucleocidin in Streptomyces calvus Is Dependent on the bldA-Specified Leu-tRNA(UUA) Molecule.

Nucleocidin is one of the very few natural products known to contain fluorine. Mysteriously, the nucleocidin producer Streptomyces calvus ATCC 13382 has not been observed to synthesize the compound since its discovery in 1956. Here, we report that complementation of S. calvus ATCC 13382 with a functional bldA-encoded Leu-tRNA(UUA) molecule restores the production of nucleocidin. Nucleocidin was detected in culture extracts by (19) F NMR spectroscopy, HPLC-ESI-MS, and HPLC-continuum source molecular absorption spectroscopy for fluorine-specific detection. The molecule was purified from a large-scale culture and definitively characterized by NMR spectroscopy and high-resolution MS. The nucleocidin biosynthetic gene cluster was identified by the presence of genes encoding the 5'-O-sulfamate moiety and confirmed by gene disruption. Two of the genes within the nucleocidin biosynthetic gene cluster contain TTA codons, thus explaining the dependence on bldA and resolving a 60-year-old mystery.

The Gene bldA, a regulator of morphological differentiation and antibiotic production in streptomyces.

Streptomyces species are well known for their particular features of morphological differentiation. On solid agar, a mold-like aerial mycelium is formed and spores are produced, in which the bld genes play a crucial role. In S. coelicolor, mutations in one specific bld gene called bldA led to a "naked" phenotype lacking aerial hyphae and spores. This peculiar behavior became a major interest for scientific research in the past and it was revealed that bldA is coding for a unique tRNA able to translate a UUA codon into the amino acid leucine. UUA codons are a very rare property of G + C-rich Streptomyces genomes. The impact of bldA on morphology can in parts be attributed to the regulatory effect of bldA on the translational level, because TTA-containing genes can only be translated into their corresponding protein in the presence of a fully functioning bldA gene. In addition to the visible effect of bldA expression on the phenotype of S. coelicolor, bldA mutants were also deficient in antibiotic production. This led to the assumption that the role of bldA must exceed translational control. Many TTA-containing genes are coding for transcriptional regulators which are activating or repressing the transcription of many more genes. Proteomics and transcriptomics are two powerful methods for identifying bldA target genes and it was possible to assign also post-translational regulation to bldA. This review wants to give a short overview on the importance of bldA as a regulator of morphological differentiation and antibiotic production by switching on "silent" gene clusters in Streptomyces.

Ruthenium- and osmium-arene complexes of 8-substituted indolo[3,2-c]quinolines: Synthesis, X-ray diffraction structures, spectroscopic properties, and antiproliferative activity.

Six novel ruthenium(II)- and osmium(II)-arene complexes with indoloquinoline modified ligands containing methyl and halo substituents in position 8 of the molecule backbone have been synthesised and comprehensively characterised by spectroscopic methods (1H, 13C NMR, UV-Vis), ESI mass spectrometry and X-ray crystallography. Binding of indoloquinolines to a metal-arene scaffold makes the products soluble enough in biological media to allow for assaying their antiproliferative activity. The complexes were tested in three human cancer cell lines, namely A549 (non-small cell lung cancer), SW480 (colon carcinoma) and CH1 (ovarian carcinoma), yielding IC50 values in the 10-6-10-7 M concentration range after continuous exposure for 96 h. Compounds with halo substituents in position 8 are more effective cytotoxic agents in vitro than the previously reported species halogenated in position 2 of the indoloquinoline backbone. High antiproliferative activity of both series of substances may be due at least in part to their potential to act as DNA intercalators.