Two mutant prion proteins expressed in cultured cells acquire biochemical properties reminiscent of the scrapie isoform.

Prion diseases are a group of fatal neurodegenerative disorders that are unique in being infectious, genetic, and sporadic in origin. Infectious cases are caused by prions, which are composed primarily of PrPSc, a posttranslationally modified isoform of the normal cellular prion protein PrPC. Inherited cases are linked to insertional or point mutations in the host gene encoding PrPC. To investigate the molecular mechanisms underlying inherited prion diseases, we have constructed stably transfected Chinese hamster ovary cells that express mouse PrPs homologous to two human PrPs associated with familial Creutzfeldt-Jakob disease. One mouse PrP molecule carries a Glu-->Lys substitution at codon 199, and the other carries an insertion of six additional octapeptide repeats between codons 51 and 90. We find that both of these mutant PrPs display several biochemical hallmarks of PrPSc when synthesized in cell culture. Unlike wild-type PrP, the mutant proteins are detergent insoluble and are relatively resistant to digestion by proteinase K, yielding an N-terminally truncated core fragment of 27-30 kDa. Pulse-chase labeling experiments demonstrate that these properties are acquired posttranslationally, and are accompanied by increased metabolic stability of the protein. Our results provide the first evidence that a molecule with properties reminiscent of PrPSc can be generated de novo in cultured cells.

Polynitrosylated proteins: characterization, bioactivity, and functional consequences.

Chemical modification of proteins is a common theme in their regulation. Nitrosylation of protein sulfhydryl groups has been shown to confer nitric oxide (NO)-like biological activities and to regulate protein functions. Several other nucleophilic side chains -- including those with hydroxyls, amines, and aromatic carbons -- are also potentially susceptible to nitrosative attack. Therefore, we examined the reactivity and functional consequences of nitros(yl)ation at a variety of nucleophilic centers in biological molecules. Chemical analysis and spectroscopic studies show that nitrosation reactions are sustained at sulfur, oxygen, nitrogen, and aromatic carbon centers, with thiols being the most reactive functionality. The exemplary protein, BSA, in the presence of a 1-, 20-, 100-, or 200-fold excess of nitrosating equivalents removes 0.6 +/- 0.2, 3.2 +/- 0.4, 18 +/- 4, and 38 +/- 10, respectively, moles of NO equivalents per mole of BSA from the reaction medium; spectroscopic evidence shows the proportionate formation of a polynitrosylated protein. Analogous reaction of tissue-type plasminogen activator yields comparable NO protein stoichiometries. Disruption of protein tertiary structure by reduction results in the preferential nitrosylation of up to 20 thus-exposed thiol groups. The polynitrosylated proteins exhibit antiplatelet and vasodilator activity that increases with the degree of nitrosation, but S-nitroso derivatives show the greatest NO-related bioactivity. Studies on enzymatic activity of tissue-type plasminogen activator show that polynitrosylation may lead to attenuated function. Moreover, the reactivity of tyrosine residues in proteins raises the possibility that NO could disrupt processes regulated by phosphorylation. Polynitrosylated proteins were found in reaction mixtures containing interferon-gamma/lipopolysaccharide-stimulated macrophages and in tracheal secretions of subjects treated with NO gas, thus suggesting their physiological relevance. In conclusion, multiple sites on proteins are susceptible to attack by nitrogen oxides. Thiol groups are preferentially modified, supporting the notion that S-nitrosylation can serve to regulate protein function. Nitrosation reactions sustained at additional nucleophilic centers may have (patho)physiological significance and suggest a facile route by which abundant NO bioactivity can be delivered to a biological system, with specificity dictated by protein substrate.

Detection of exocytosis at individual pancreatic beta cells by amperometry at a chemically modified microelectrode.

Amperometry at a carbon fiber microelectrode modified with a composite of ruthenium oxide and cyanoruthenate was used to monitor chemical secretions of single pancreatic beta cells from rats and humans. When the insulin secretagogues glucose, tolbutamide, and K+ were applied to the cell, a series of randomly occurring current spikes was observed. The current spikes were shown to be due to the detection of chemical substances secreted from the cell. Chromatography showed that the primary secreted substance detected by the electrode was insulin. The current spikes were strongly dependent on external Ca2+, had an average area that was independent of the stimulation method, and had an area distribution which corresponded to the distribution of vesicle sizes in beta cells. It was concluded that the spikes were due to the detection of concentration pulses of insulin secreted by exocytosis.

Yeast synaptobrevin homologs are modified posttranslationally by the addition of palmitate.

Yeast possess two homologs of the synaptobrevin family of vesicle-associated membrane proteins that function in membrane recognition and vesicle fusion. Yeast proteins Snc1 and Snc2 localize to secretory vesicles and are required for constitutive exocytosis. They also form a physical complex with a plasma membrane protein, Sec9, which is necessary for vesicle docking and fusion to occur in vivo. Formation of this molecular complex, as a prerequisite for vesicle fusion, appears to have been conserved evolutionarily. Here we demonstrate that Snc proteins undergo a single posttranslational modification with the addition of a palmitate moiety to Cys-95 in Snc1. Modification of Cys-95 (which is located proximal to the transmembrane domain) is rapid, occurs in the endoplasmic reticulum, and is long-lasting. Mutation of Cys-95 to Ser-95 blocks palmitoylation and appears to affect Snc protein stability. This provides evidence that synaptobrevin-like proteins are modified posttranslationally, and we predict that fatty acylation may be common to those found in higher eukaryotes.

Specific and reversible immobilization of proteins tagged to the affinity polypeptide C-LytA on functionalized graphite electrodes

We have developed a general method for the specific and reversible immobilization of proteins fused to the choline-binding module C-LytA on functionalized graphite electrodes. Graphite electrode surfaces were modified by diazonium chemistry to introduce carboxylic groups that were subsequently used to anchor mixed self-assembled monolayers consisting of N,N-diethylethylenediamine groups, acting as choline analogs, and ethanolamine groups as spacers. The ability of the prepared electrodes to specifically bind C-LytA-tagged recombinant proteins was tested with a C-LytA-β-galactosidase fusion protein. The binding, activity and stability of the immobilized protein was evaluated by electrochemically monitoring the formation of an electroactive product in the enzymatic hydrolysis of the synthetic substrate 4-aminophenyl β-D-galactopyranoside. The hybrid protein was immobilized in an specific and reversible way, while retaining the catalytic activity. Moreover, these functionalized electrodes were shown to be highly stable and reusable. The method developed here can be envisaged as a general, immobilization procedure on the protein biosensor field.

Afffinity tag excision strategies for Car9-tagged proteins

Thesis (Master's)--University of Washington, 2016-06

tRNASer(CGA) differentially regulates expression of wild-type and codon-modified papillomavirus L1 genes

Exogenous transfer RNAs (tRNAs) favor translation of bovine papillomavirus 1 wild-type (wt) L1 mRNA in in vitro translation systems (Zhou et al. 1999, J. Virol., 73, 4972-4982). We, therefore, investigated whether papillomavirus (PV) wt L1 protein expression could be enhanced in eukaryotic cells following exogenous tRNA supplementation. Both Chinese hamster ovary (CHO) and Cos1 cells, transfected with PV1 wt L1 genes, effectively transcribed the genes but did not translate them. However, L1 protein translation was demonstrated following co-transfection with the L1 gene and a gene expressing tRNA(Ser)(CGA). Cell lines, stably transfected with a bovine papillomavirus 1 (BPV1) wt L1 expression construct, produced L1 protein after the transfection of the tRNA(Ser)(CGA) gene, but not following the transfection with basal vectors, suggesting that tRNA(Ser)(CGA) gene enhanced wt L1 translation as a result of endogenous tRNA alterations and phosphorylation of translation initiation factors elF4E and elF2alpha in the tRNA(Ser)(CGA) transfected L1 cell lines. The tRNA(Ser)(CGA) gene expression significantly reduced translation of L1 proteins expressed from codon-modified (HB) PV L1 genes utilizing mammalian preferred codons, but had variable effects on translation of green fluorescent proteins (GFPs) expressed from six serine GFP variants. The changes of tRNA pools appear to match the codon composition of PV wt and HB L1 genes and serine GFP variants to regulate translation of their mRNAs. These findings demonstrate for the first time in eukaryotic cells that translation of the target genes can be differentially influenced by the provision of a single tRNA expression construct.

A recombinant diheme SoxAX cytochrome - Implications for the relationship between EPR signals and modified heme-ligands

The multiheme SoxAX proteins are notable for their unusual heme ligation (His/Cys-persulfide in the SoxA subunit) and the complexity of their EPR spectra. The diheme SoxAX protein from Starkeya novella has been expressed using Rhodobacter capsulatus as a host expression system. rSoxAX was correctly formed in the periplasm of the host and contained heme c in similar amounts as the native SoxAX. ESI-MS showed that the full length rSoxA, in spite of never having undergone catalytic turnover, existed in several forms, with the two major forms having masses of 28 687 +/- 4 and 28 718 +/- 4 Da. The latter form exceeds the expected mass of rSoxA by 31 4 Da, a mass close to that of a sulfur atom and indicating that a fraction of the recombinant protein contains a cysteine persulfide modification. EPR spectra of rSoxAX contained all four heme-dependent EPR signals (LS1a, LS1b, LS2, LS3) found in the native SoxAX proteins isolated from bacteria grown under sulfur chemolithotrophic conditions. Exposure of the recombinant SoxAX to different sulfur compounds lead to changes in the SoxA mass profile as determined by ESI while maintaining a fully oxidized SoxAX visible spectrum. Thiosulfate, the proposed SoxAX substrate, did not cause any mass changes while after exposure to dimethylsulfoxide a + 112 +/- 4 Da form of SoxA became dominant in the mass spectrum. (c) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Mass spectrometric analysis of HOCl- and free-radical-induced damage to lipids and proteins

In inflammatory diseases, release of oxidants leads to oxidative damage to biomolecules. HOCl (hypochlorous acid), released by the myeloperoxidase/H2O2/Cl- system, can cause formation of phospholipid chlorohydrins, or alpha-chloro-fatty aldehydes from plasmalogens. It can attack several amino acid residues in proteins, causing post-translational oxidative modifications of proteins, but the formation of 3-chlorotyrosine is one of the most stable markers of HOCl-induced damage. Soft-ionization MS has proved invaluable for detecting the occurrence of oxidative modifications to both phospholipids and proteins, and characterizing the products generated by HOCl-induced attack. For both phospholipids and proteins, the application of advanced mass spectrometric methods such as product or precursor ion scanning and neutral loss analysis can yield information both about the specific nature of the oxidative modification and the biomolecule modified. The ideal is to be able to apply these methods to complex biological or clinical samples, to determine the site-specific modifications of particular cellular components. This is important for understanding disease mechanisms and offers potential for development of novel biomarkers of inflammatory diseases. In the present paper, we review some of the progress that has been made towards this goal.

Transglutaminase mediated grafting of silk proteins onto wool fabrics leading to improved physical and mechanical properties

Transglutaminases have the ability to incorporate primary amines and to graft peptides (containing glutamine or lysine residues) into proteins. These properties enable transglutaminases to be used in the grafting of a range of compounds including peptides and/or proteins onto wool fibres, altering their functionality. In this paper we investigated the transglutaminase mediated grafting of silk proteins into wool and its effect on wool properties. A commercial hydrolysed silk preparation was compared with silk sericin. The silk sericin protein was labelled with a fluorescent probe which was used to demonstrate the efficiency of the TGase grafting of such proteins into wool fibres. The TGase mediated grafting of these proteins led to a significant effect on the properties of wool yarn and fabric, resulting in increased bursting strength, as well as reduced levels of felting shrinkage and improved fabric softness. Also observed was an accumulation of deposits on the surface of the treated wool fibres when monitored by SEM and alterations in the thermal behaviour of the modified fibres, in particular for mTGase/sericin treated fibres which, with the confocal studies, corroborate the physical changes observed on the treated wool fabric. © 2006 Elsevier Inc. All rights reserved.

Novel pMDI formulations for pulmonary delivery of proteins

In this paper, we demonstrate that co-spray-drying a model protein with sodium carboxymethylcellulose (NaCMC) protects protein integrity during spray-drying, and that the resultant spray-dried powders can be successfully dispersed in hydrofluoroalkane (HFA) propellant to prepare pressurised metered dose (pMDI) formulations that exhibit high respirable fractions. The spray-dried powders were formulated as HFA-134a pMDI suspensions in the absence of any other excipients (e.g. surfactants) or co-solvents (e.g. ethanol). The in vitro aerosolisation profile of these systems was assessed using the twin stage impinger; fine particle fractions (FPF) ≥50% of the recovered dose were obtained. Following storage for five months, the aerosolisation performance was reassessed; the NaCMC-free formulation demonstrated a significant decrease in FPF, whereas the performance of the NaCMC-modified formulations was statistically equivalent to their initial performance. Thus, formulation of pMDI suspensions using NaCMC-based spray-dried powders is a promising approach for the pulmonary delivery of proteins and peptides. © 2009 Elsevier B.V. All rights reserved.

The use of sodium carboxymethylcellulose in the preparation of spray-dried proteins for pulmonary drug delivery

The use of sodium carboxymethylcellulose (NaCMC) as a spray-drying excipient in the preparation of inhalable formulations of proteins was investigated, using alkaline phosphatase as a model functional protein. Two spray-dried powders were investigated: a control powder comprising 100% (w/w) alkaline phosphatase and a test powder comprising 67% (w/w) NaCMC and 33% (w/w) alkaline phosphatase. Following physicochemical characterisation, the powders were prepared as both dry powder inhaler (DPI) and pressurised metered dose inhaler (pMDI) formulations. The aerosolisation performance of the formulations was assessed using a Multi-Stage Liquid Impinger, both immediately after preparation and over a 16-week storage period. Formulating the control powder as a DPI resulted in a poor fine particle fraction (FPF: 10%), whereas the FPF of the NaCMC-modified DPI formulation was significantly greater (47%). When the powders were formulated as pMDI systems, the control and NaCMC-modified powders demonstrated FPFs of 52% and 55%, respectively. Following storage, reduced FPF was observed for all formulations except the NaCMC-modified pMDI system; the performance of this formulation following storage was statistically equivalent to that immediately following preparation. Co-spray-drying proteins and peptides with NaCMC may therefore offer an alternative method for the preparation of stable and respirable pMDI formulations for pulmonary delivery. © 2010 Elsevier B.V.