http://www.springerprotocols.com/cdp/access/showWebFeedListing This feed provides the latest 25 protocols in the given category. http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-234-2_1 From an early stage of prion research, tissue cultures that could support and propagate the scrapie agent were sought after. The earliest attempts were explants from brains of infected mice, and their growth and morphological characteristics were compared with those from uninfected mice (1). Using the explant technique, several investigators reported increased cell growth in cultures established from scrapie-sick brain compared with cultures from normal mice (1, 2). These are odd findings in the light of the massive neuronal cell death known to occur in scrapie-infected brains; however, the cell types responsible for the increased cell growth in the scrapie-explants most probably were not neuronal. The first successful cell culture established in this way, in which the scrapie agent was serially and continuously passaged beyond the initial explant, was in the scrapie mouse brain culture (3), which is still used today (4, 5). This chapter describes the generation and use of chronically prion-infected cell lines as cell culture models of prion diseases. These cell lines have been crucial for the current understanding of the cell biology of both the normal (PrPC) and the pathogenic isoform (PrPSc) of the prion protein. They also have been useful in the development of antiprion drugs, prospectively used for therapy of prion diseases, and they offer an alternative approach for transmission/infectivity assays normally performed by mouse bioassay. Cell culture models also have been used to study prion-induced cytopathological changes, which could explain the typical spongiform neurodegeneration in prion diseases. 2008-06-04T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-234-2_10 Certain applications in the prion field require recombinant prion protein (PrP) of high purity and quality. Here, we report an experimental procedure for expression and purification of full-length mammalian prion protein. This protocol has been proved to yield PrP of extremely high purity that lacks PrP adducts, which are normally generated as a result of spontaneous oxidation or degradation. 2008-06-04T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-234-2_11 The availability of recombinant prion proteins (recPrP) has been exploited as a model system to study PrP-mediated toxicity, conversion, and infectivity. According to the protein only hypothesis, the central event in the pathogenesis of prion diseases is the conversion of PrPC to PrPSc. This involves a dramatic increase in β sheet conformation as PrPC is converted to PrPSc, and it is widely believed that this conformational change affects the as-yet undefined function of PrPC. Although there are many methods available to monitor for the changes in the structural makeup of PrP mutants and oligomers formed with respect to disease relevance, circular dichroism is one of the most popular methods used. In this chapter, we discuss the fundamental principles of circular dichroism and its current role and applications in prion disease research. 2008-06-04T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-234-2_12 The prion protein (PrP) is the key protein implicated in diseases known as transmissible spongiform encephalopathies. PrP has been shown to be a metallo-protein that binds copper (Cu), and copper might have a role in the normal function of the protein. Conversely, PrP expression in yeast led us to suggest that the protein might be involved in the regulation of Cu homeostasis. In the presence of excess Cu in the growth medium, PrP expression limited the increase of the total number of Cu atoms per cell to a maximum of 14-fold compared with mock control cells, which showed a 52-fold increased intracellular Cu level. Conclusively, we suggest that PrP expression itself has a regulatory or buffering function for the cellular Cu level in yeast cells, most likely due to binding of Cu to the multiple Cu binding sites. 2008-06-04T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-234-2_13 The binding of paramagnetic metal ions is thought to be an essential function of the prion protein and lends itself to interrogation by electron paramagnetic resonance (EPR), which probes the local coordination environment of bound metal ions to provide details of the metal-binding affinity, stoichiometry, and the symmetry and identity of its ligating atoms. It is also capable of identifying reactive oxygen/nitrogen species and peptide-derived radicals, in addition to monitoring protein-membrane dynamics and conformation by using site-directed spin labeling. An overview of the EPR technique as applied to the prion protein is given, key results are summarized, and some future experimental avenues are outlined. 2008-06-04T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-234-2_14 Human prion diseases are associated with a range of clinical presentations, and they are classified by both clinicopathological syndrome and etiology, with subclassification according to molecular criteria. Here, we describe procedures that are used within the MRC Prion Unit to determine a molecular diagnosis of human prion disease. Sequencing of the PRNP open reading frame to establish the presence of pathogenic mutations is described, together with detailed methods for immunoblot or immunohistochemical determination of the presence of abnormal prion protein in brain or peripheral tissues. 2008-06-04T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-234-2_15 Numerous transmembrane and glycosylphosphatidylinositol (GPI)-anchored proteins, covering a vast range of structural and functional classes, are recognized to undergo proteolytic cleavage or shedding from the plasma membrane. Although this widespread phenomenon seems fundamental to normal cellular biology, proteolytic processing also seems to play a central role in the pathogenesis of some neurodegenerative disorders such as Alzheimer's disease. An analogous situation may exist in prion disorders. The GPI-anchored cellular prion protein (PrPC) may be endoproteolytically cleaved at two different sites: one at the C-terminal end of the octameric repeat region and the other within a potentially neurotoxic and amyloidogenic region of the protein. The relevance of these alternative proteolytic events to normal cell function and pathogenesis is incompletely resolved. Study and characterization of the constitutive processing of PrPC will provide insight into the biological relevance of alternative cleavages in terms of normal PrPC function, and also into the potential role, if any, to disease causation. 2008-06-04T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-234-2_16 Within the spectrum of sporadic human transmissible spongiform encephalopathies (TSEs), there is considerable diversity of disease phenotypes. At least part of this variation is thought to be on the basis of different “strains” of prions (the infectious agent). Tissue deposition of PrPres (the abnormal disease-associated conformation of the prion protein) is considered a hallmark of TSE pathology, and it can be visualized by Western blotting typically as three bands depicting the diglycosylated, monoglycosylated, and unglycosylated species. It is the mobility of the unglycosylated PrPres, and the relative abundance of the two glycosylated bands, along with the prion protein gene (PRNP) codon 129 genotype, that seem to correlate with distinct clinico-pathological profiles of sporadic Creutzfeldt-Jakob disease. 2008-06-04T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-234-2_17 Prions represent a new biological paradigm of protein-mediated information transfer. In mammals, prions are the cause of fatal, transmissible neurodegenerative diseases, often referred to as transmissible spongiform encephalopathies. Many unresolved issues remain, including the exact molecular nature of the prion, the detailed mechanism of prion propagation, and the mechanism by which prion diseases can be both genetic and infectious. In addition, we know little about the mechanism by which neurons degenerate during prion diseases. Tied to this, the physiological function of the normal form of the prion protein remains unclear, and it is uncertain whether loss of this function contributes to prion pathogenesis. The factors governing the transmission of prions between species remain unclear, in particular the means by which prion strains and PrP primary structure interact to affect interspecies prion transmission. Despite all these unknowns, dramatic advances in our understanding of prions have occurred because of their transmissibility to experimental animals and the development of transgenic mouse models has done much to further our understanding about various aspects of prion biology. In this chapter, I review recent advances in our understanding of prion biology that derive from this powerful and informative approach. 2008-06-04T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-234-2_18 The unconventional nature of the infectious agent of prion diseases poses a challenge to conventional infection control methodologies. The extra neural tissue distribution of variant and sporadic Creutzfeldt—Jakob disease has increased concern regarding the risk of prion disease transmission via general surgical procedures and highlighted the need for decontamination procedures that can be incorporated into routine processing. This chapter describe a quantitative method for assessing the prionocidal activity of chemical and physical decontamination methods against surface-bound prion infectivity. 2008-06-04T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-234-2_2 Prion protein (PrP)C expression levels and protein localization are known to be affected by factors such as metal ions and oxidative stress. By the development of a green fluorescent protein (GFP)-PrPC fusion protein, the movement of PrP can be followed in real time. Furthermore, alterations in cellular metabolism can be detected while cells are still viable. The internalization response of PrP to 20 μM manganese (Mn) in divalent metal ion-depleted media is used to demonstrate the movement of GFP-tagged proteins in live cells and real tim0e. A live cell microtiter plate assay shows that PrP null cells are less capable of dealing with Mn-induced oxidative stress. In addition, this chapter outlines several complementary techniques for studying live cells and GFP fusion proteins. 2008-06-04T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-234-2_3 Prion infectivity is often linked to presence of the protease-resistant isoform of prion protein (PrP), PrPres; therefore, it is of highest interest to have convenient methods for rapid detection of PrPres in the research laboratory. For detection of PrPres in model systems to confirm infectivity, there are several methods that can be applied. This chapter focuses on detection of PrPres by proteinase K digestion followed by Western blot, which is the only method that is both quantitative and qualitative. For large-scale screening of PrPres content in samples, the dot blot method offers a great advantage for detecting PrPres, and this method is also thoroughly described in this chapter. 2008-06-04T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-234-2_4 Prions are usually quantified by bioassays based on intracerebral inoculation of animals, which are slow, imprecise, and costly. We have developed a cell-based prion assay that is based on the isolation of cell lines highly susceptible to certain strains (Rocky Mountain Laboratory and 22L) of mouse prions and a method for identifying individual, prion-infected cells and quantifying them. In the standard scrapie cell assay (SSCA), susceptible cells are exposed to prion-containing samples for 4 days, grown to confluence, passaged two or three times, and the proportion of rPrPSc-containing cells is determined with automated counting equipment. The dose response is dynamic over 2 logs of prion concentrations. The SSCA has a standard error of ±20–30%, is as sensitive as the mouse bioassay, 10 times faster, at least 2 orders of magnitude less expensive, and it is suitable for robotization. Assays performed in a more time-consuming end point titration format extend the sensitivity and show that infectivity titers measured in tissue culture and in the mouse are similar. 2008-06-04T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-234-2_5 Prion-propagating cell lines are an efficient and useful means for studying the cellular and molecular mechanisms implicated in prion disease. Use of cell-based models has lead to the finding that prion protein (PrPC) and PrPSc are released from cells in association with exosomes. Furthermore, exosomes have been shown to act as vehicles for infectivity, transferring PrPSc between cell lines and providing a mechanism for prion spread between tissues. As a role for exo-somes in prion disease is emerging, this chapter outlines a method for the generation of prion-infected cell lines and the isolation and characterization of PrPC- and PrPSc-containing exosomes. 2008-06-04T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-234-2_6 Prion peptide (PrP) neurotoxicity has been modelled in vitro by using synthetic peptides derived from the PrPC sequence. The major region of neurotoxicity has been localized to the hydrophobic domain located in the middle of the PrP sequence. The neurotoxicity assays are typically performed on cultured mouse cerebellar neurons derived from neonatal pups, and viability can be monitored by a variety of assays, including MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium); MTS (3-(4,5-dimeth-ylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) lactate dehydrogenase release; and apoptotic assays. These neurotoxicity studies have been useful in identifying cofactors, such as PrPC and metals as modulators of PrP peptide-mediated neurotoxicity. Given the biosafety issues associated with handling and purifying infectious prions, the use of synthetic peptides that display a dependence upon PrPC expression for toxicity, as per the PrPSc agent for infectivity, supports the relevance of using these synthetic peptides for understanding PrP-mediated neurotoxicity. 2008-06-04T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-234-2_7 A central event in the transmission and pathogenesis of transmissible spongiform encephalopathy diseases is the misfolding of the prion protein. Considerable progress has been made in our understanding of this misfolding event through the development of cell-free assays that mimic the molecular features of prion propagation. This chapter reviews the contribution of cell-free assays to our understanding of prion propagation. 2008-06-04T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-234-2_8 Misfolding and aggregation of prion protein (PrP) is related to several neurodegenerative diseases in humans such as Creutzfeldt—Jacob disease, fatal familial insomnia, and Gerstmann—Straussler—Sheinker disease. Amyloid fibrils prepared from recombinant PrP in vitro share many features of the infectious prions. These fibrils can be used as a synthetic surrogate of PrPSc for development of prion diagnostics, including generation of PrPSc-specific antibody, for screening of antiprion drugs, or for development of antiprion decontamination procedures. Here, we describe the methods of preparation of prion protein fibrils in vitro and biochemical assays for assessing physical properties and the quality of fibrils. 2008-06-04T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-234-2_9 The infectious agents of prion diseases are unorthodox, and they seem to be composed primarily of a misfolded glycoprotein called the prion protein (PrP). Replication of prion infectivity is associated with the conversion of PrP from its normal, cellular form (PrPC) into a pathogenic form (PrPSc), which is characterized biochemically by relative detergent insolubility and protease resistance. Several techniques have been developed in which PrPC molecules can be converted into the PrPSc conformation in vitro (1–8). These biochemical techniques recapitulate several specific aspects of in vivo prion propagation (1–3), and one method, the protein misfolding cyclic amplification technique, also has been shown to amplify infectivity (5). In this chapter, we describe a method for amplifying PrPSc molecules from hamster prions in vitro using purified substrates. Specific protocols for substrate preparation, reaction mixture, and product detection are explained. Purified PrPSc amplification assays are currently being used to study the biochemical mechanism of prion formation. 2008-06-04T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-084-7_1 Oxidation of sulfhydryl groups to form a disulfi de bond is one of the most common post-translational modifi cations in proteins. Disulfi de bonds play important roles in stabilizing three-dimensional structure and modulating bioactivity of the cystinyl proteins. The determination of disulfi de bond linkage is therefore an integral part of structural characterization of proteins. A mass spectrometry-based strategy utilizing chemical cleavage at cysteine residues following cyanylation reaction is described for the identifi cation of both sulfhydryl and disulfi de bond linkage in proteins. The method has been particularly powerful for the assignment of disulfi de bonds in proteins containing adjacent or closely spaced cysteines. 2008-04-04T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-084-7_10 ISG15 is a ubiquitin-like modifi er that is conjugated to target proteins by a sequential reaction catalyzed by E1/E2/E3 enzymes (protein ISGylation). ISG15 and protein ISGylation are upregulated by interferon stimuli. ISG15 functions as an antiviral protein against Sindbis virus and HIV-1, but the molecular mechanism remains unknown. Here we describe in detail methods for detecting and analyzing protein ISGylation. The methods consist of plasmid transfection and affi nity purifi cation of ISGylated proteins. In addition, we describe a method for detecting ISGylation of a target protein, Ubc13. 2008-04-04T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-084-7_11 A wide variety of post-translational modifications of expressed proteins are known to occur in living organisms (1). Although their presence in an organism cannot be predicted from the genome, these modifications can play critical roles in protein structure and function. The identification of post-translational modifications can be critical in understanding the functions of proteins involved in important biological pathways and mass spectrometry offers a fast, accurate method for observing them. This chapter describes the procedure for analyzing ribosomal proteins of Escherichia coli by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and Caulobacter crescentus ribosomal proteins by electrospray quadrupole time-of-flight (ESI-QTOF) mass spectrometry. 2008-04-04T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-084-7_12 Palmitoylation or S-acylation is the post-translational attachment of fatty acids to cysteine residues and is common among integral and peripheral mem brane proteins. Palmitoylated proteins have been found in every eukaryotic cell type examined (yeast, insect, and vertebrate cells), as well as in viruses grown in these cells. The exact functions of protein palmitoylation are not well understood. Intrin sically hydrophilic proteins, especially signaling molecules, are anchored by long chain fatty acids to the cytoplasmic face of the plasma membrane. Palmitoylation may also promote targeting to membrane subdomains enriched in glycosphingolip ids and cholesterol or affect protein–protein interactions. 2008-04-04T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-084-7_13 Glycosylphosphatidylinositol (GPI) is a complex glycolipid structure that acts as a membrane anchor for many cell-surface proteins of eukaryotes. GPI-anchored proteins are particularly abundant in protozoa and represent the major carbohydrate modification of many cell-surface parasite proteins. A minimal GPI-anchor precursor consists of core glycan (ethanolamine-P-Manα1–2Manα1–6Manα1–4GlcNH2) linked to the 6-position of the D-myo-inositol ring of phos-phatidylinositol. Although the GPI core glycan is conserved in all organisms, many differences in additional modifications to GPI structures and biosynthetic pathways have been reported. The preassembled GPI-anchor precursor is post-translationally transferred to a variety of membrane proteins in the lumen of the endoplasmic reticulum in a transamidase-like reaction during which a C-terminal GPI attachment signal is released. Increasing evidence show that a significant proportion of the synthesized GPIs are not used for protein anchoring, particularly in protozoa in which a large amount of free GPIs are being displayed at the cell surface. The characteristics of GPI biosynthesis are currently being explored for the development of parasite-specific inhibitors. Especially as this pathway, at least for Trypanosoma brucei, has been validated as a drug target. 2008-04-04T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-084-7_14 The detailed characterization of complex protein mixtures as in samples from biological sources cannot be sufficiently performed by separation of polypeptides according to their molecular weight as is done by conventional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (1DE). For analysis of such samples, 2-dimensional gel electrophoresis (2DE) is the preferable methodological approach because it combines separation of polypeptides according to isoelectric properties and molecular weight as well. The resulting pattern of protein spots does not only provide information on composition of samples because of the complexity of a mixture of polypeptides. It delivers also a picture on the microheterogenity of polypeptides caused by post-translational modifications. These might be of natural or artificial type and occur during biosynthetic processing of a polypeptide or within industrial scale production. The presented method describes an experimental approach to investigate the influence of glycosylation in general and sialylation exclusively on spot pattern of proteins separated by 2DE. 2008-04-04T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-084-7_15 Oligosaccharides in glycoproteins by their very nature infl uence many aspects of protein function, e.g., half-life and activity/potency. Recombinant IgGs constitute a major portion of therapeutic proteins. Though the glycans in IgGs account for about 2% of the total weight, they influence biologic activity apart from antigen binding. Characterization of the carbohydrates is not only a regulatory requirement but it may allow understanding of structure-function of proteins. Current advances in analytical techniques permit structural elucidation of small quantities of glycoproteins. At a fi rst glance monosaccharide analysis may provide insight into the types of glycosylation similar to information afforded by amino acid composition. It is the only stand-alone technique by which individual sugar residues can be identifi ed and quantitated (mol/mol). Fluorescent anthranilic acid (AA) has been extensively used as a high sensitivity detection tag for carbohydrates. HPLC methods with fl uorescence detection described in this chapter are suitable for the analysis of monosaccharides (including sialic acids) on a routine basis. AA is used for the determination of hexoses and hexosamines, and o-phenylenediamine for sialic acids. These methods were validated and found to be highly reproducible compared to HPAEC-PAD and CE methods. 2008-04-04T04:00:00Z