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Kinetic analysis of FlAsHPrP(230TC) identifies a new metabolic fragment of PrP. (A) Structure of PrP(230TC). The GPI-attachment signal is removed from the nascent protein before GPI anchor addition to Ser 230. (B) Summary of metabolic fragments of FlAsH-PrP(230TC). Proteolytic fragments observed were generated by endogenous proteases. The fluorescent gel shows FlAsHPrP(230TC) fragments in PTA precipitates from 22L(230TC) cells chased for several hours after labeling. To create this specific image for reference purposes only, the precipitate was not washed with N-lauroylsarcosine buffer after PTA precipitation to retain contaminant fragments that are preferentially reduced by washing (e.g., full-length). (C) Nontransfected N2a cells have a C3-like fragment. Immunoblotting of wild-type PrP from detergent phase of TX114 lysates of nontransfected N2a cells with monoclonal R1 antibody, which recognizes C-terminus (residues 220231) of PrP. Arrowhead indicates the 9-kDa C3-like fragment. (D) Kinetics of FlAsHPrP(230TC) in 22L(230TC) cells. The fluorescent gels and the graphs show the results of FlAsH-pulse-chase analysis of 22L(230TC) cells. Lysate fractions correspond to 1/20 the cell equivalents loaded in the PTA fractions. The experiments were performed in quadruplicate and the curves show average band intensity SEM. The arrows indicate full-length FlAsH-PrP(230TC), the arrowhead indicates the 21-kDa truncated PrPres band, and the asterisk indicates the C3 fragment. Error bars are included on all graphs but in many cases are small enough to be obscured by the plot symbols. Lysate, kinetics of FlAsH-PrP(230TC) in cell lysate samples. PTA, kinetics of FlAsHPrP(230TC) in PTA-precipitated samples. f, Full, full-length FlAsH-PrP(230TC). , C3, C3 fragment. OE, 21 kDa, 21-kDa band. , Chy-res, samples digested with chymotrypsin.
Source publication
Fluorescent tagging is a powerful tool for imaging proteins in living cells. However, the steric effects imposed by fluorescent tags impair the behavior of many proteins. Here, we report a novel technique, Instant with DTT, EDT, And Low temperature (IDEAL)-labeling, for rapid and specific FlAsH-labeling of tetracysteine-tagged cell surface proteins...
Contexts in source publication
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... time, full-length FlAsH-PrP initially increased and then decreased quickly; reciprocally, the 21-kDa fragment appeared and increased ( Figure 2F, PTA). The reciprocal nature of the kinetics of full-length FlAsH-PrP versus the 21-kDa fragment was consistent with the formation and subsequent truncation of FlAsH-PrPres by endogenous cys- teine proteases (Supplemental Figure 3 and see below). These kinetics suggested N-terminal truncation of FlAsH- PrPres occurs shortly after conversion. ...
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... explore the flexibility to insert the TC motif at desired locations within a target protein, we created a PrP construct [PrP(230TC)] with the TC-motif adjacent to the GPI attach- ment site ( Figure 3A). This construct enabled the detection of C-terminal products of PrP metabolism. ...
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... construct enabled the detection of C-terminal products of PrP metabolism. Three FlAsH- labeled fragments were common to both uninfected ( Figure 1A, lane 8) and scrapie-infected (Figure 3, B and D) PrP(230TC)-expressing cells. These consisted of full-length PrP; a fragment of 17-18 kDa, likely a proteolytic product called C1 (Chen et al., 1995;Taraboulos et al., 1995); and a previously unreported fragment of 9 kDa, which we called C3 ( Figure 3B). ...
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... FlAsH- labeled fragments were common to both uninfected ( Figure 1A, lane 8) and scrapie-infected (Figure 3, B and D) PrP(230TC)-expressing cells. These consisted of full-length PrP; a fragment of 17-18 kDa, likely a proteolytic product called C1 (Chen et al., 1995;Taraboulos et al., 1995); and a previously unreported fragment of 9 kDa, which we called C3 ( Figure 3B). A 21-kDa fragment was specific to scrapie-infected cells and corresponded to truncated FlAsH-PrPres (see below). ...
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... 21-kDa fragment was specific to scrapie-infected cells and corresponded to truncated FlAsH-PrPres (see below). A C3-like fragment was present in wild-type N2a cells, providing evidence that this newly identified fragment was not an artifact of FlAsH-PrP(230TC) ( Figure 3C). ...
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... we determined the kinetics of FlAsH-PrP(230TC) metabolism. Full-length FlAsH-PrPsen(230TC) showed a half-life of 4 h ( Figure 3D, lysate). The C3 fragment accu- mulated during the chase period ( Figure 3D, asterisk, ly- sate). ...
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... FlAsH-PrPsen(230TC) showed a half-life of 4 h ( Figure 3D, lysate). The C3 fragment accu- mulated during the chase period ( Figure 3D, asterisk, ly- sate). In PTA precipitates with and without chymotrypsin digestion to monitor the kinetics of FlAsH-PrP(230TC) con- version to FlAsH-PrPres, the kinetics of FlAsH-PrP(230TC) basically mirrored that of FlAsH-PrP(90TC) ( Figure 3D). ...
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... C3 fragment accu- mulated during the chase period ( Figure 3D, asterisk, ly- sate). In PTA precipitates with and without chymotrypsin digestion to monitor the kinetics of FlAsH-PrP(230TC) con- version to FlAsH-PrPres, the kinetics of FlAsH-PrP(230TC) basically mirrored that of FlAsH-PrP(90TC) ( Figure 3D). The estimated conversion efficiency for FlAsH-PrP(230TC) was somewhat lower than FlAsH-PrP(90TC) (1-2%). ...
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... kinetics of FlAsH- PrP(230TC) in PTA-precipitated samples (lanes 12-24). Ch, samples digested with chymotrypsin (lanes 23-24). Full, full-length FlAsH- PrP(230TC). ...
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... increase in full-length FlAsH-PrP was similar between treated and untreated cells, but the 21-kDa truncated FlAsH- PrPres fragment did not occur in E64-treated samples even with a 6-h chase ( Figure 4B, lanes 12-22). Surprisingly, the absence of the 21-kDa band was actually due to inhibition of the N-terminal truncation of FlAsH-PrPres by endogenous cysteine proteases rather than inhibition of FlAsH-PrPres biosynthesis as shown by analysis of chymotrypsin-digested samples ( Figure 4B, arrowhead, lane 23 vs. lane 24). It is notable that treatment with E64 or leupeptin ( Figure 4B, lane 22 and Supplemental Figure 3) gave rise to a new band of slightly lower apparent MW than full-length PrP, suggesting that proteases other than cysteine proteases may also par- ticipate in the processing of nascent FlAsH-PrPres. ...
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... the absence of the 21-kDa band was actually due to inhibition of the N-terminal truncation of FlAsH-PrPres by endogenous cysteine proteases rather than inhibition of FlAsH-PrPres biosynthesis as shown by analysis of chymotrypsin-digested samples ( Figure 4B, arrowhead, lane 23 vs. lane 24). It is notable that treatment with E64 or leupeptin ( Figure 4B, lane 22 and Supplemental Figure 3) gave rise to a new band of slightly lower apparent MW than full-length PrP, suggesting that proteases other than cysteine proteases may also par- ticipate in the processing of nascent FlAsH-PrPres. Because FlAsH-PrPres levels reach a plateau within 5.5-6 h after labeling ( Figure 3D, arrowhead, lane 17 vs. ...
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... provide an example of correlative biochemical and microscopy analysis of a TC-tagged protein, we visualized the endocytosis of AF568-FlAsH-labeled PrP(230TC) by live cell time lapse confocal microscopy at hourly time points comparable with those in Figure 3D (lysate). At the first time point (0 h), the majority of PrP(230TC) was present at the cell surface, although a small population localized to perinuclear vesicles also was observed ( Figure 5E). ...
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... the present study, PrPres biosyn- thesis was not inhibited by E64 even at concentrations that blocked complete N-terminal truncation of newly formed PrPres, suggesting that the anti-prion effects of E64 are in- dependent of its effects on PrPres proteolysis. Interestingly, we did observe evidence of a very small N-terminal trunca- tion of FlAsH-PrPres formed in the presence of cysteine protease inhibitors (Supplemental Figure 3), but the pro- tease(s) involved remains to be identified. Our data are consistent with a time dependence to the inhibitory effect of E64, raising the possibility that cysteine proteases may indi- rectly contribute to PrPres propagation via the modification of other cellular factors analogous to the effects of a partic-ular tyrosine kinase (Ertmer et al., 2004). ...
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The metacaspase Mca1 from Saccharomyces cerevisiae displays a Q/N-rich region at its N-terminus reminiscent of yeast prion proteins. In this study, we show that the ability of Mca1 to form insoluble aggregates is modulated by a peptide stretch preceding its putative prion-forming domain. Based on its genomic locus, three potential translational sta...
Prion Proteins (PrP) from different species have the ability to tightly bind Cu2+ ions. Copper coordination sites are located at the disordered and flexible N-terminal region which contains several His anchoring sites. Among them, two His residues are found in the so called amyloidogenic PrP region which is believed to play a key role in the proces...
Prions are self-propagating infectious protein aggregates of mammals and fungi. The exact mechanism of prion formation is poorly understood. In a recent study, a comparative analysis of the aggregation propensities of chimeric proteins derived from the yeast Sup35p and mouse PrP prion proteins was performed in neuroblastoma cells. The cytosolic exp...
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Prion diseases are neurodegenerative disorders that affect many mammalian species. Mammalian prion proteins (PrPs) can misfold into many different aggregates. However, only a small subpopulation of these structures is infectious. One of the major unresolved questions in prion research is identifying which specific structural features of these misfo...
Citations
... While, on the one hand, it might confer protection against prion diseases as the reduction of PrP C at the cellular surface would reduce the substrate for PrP C -to-PrP Sc conversion, and shed PrP C may impair this misfolding by binding to extracellular prion seeds (37)(38)(39)(40); on the other hand, it could facilitate prion spread and plaque formation by generating anchorless diffusible prions (32,(41)(42)(43)(44)(45)(46). Finally, a newly discovered endoproteolytic processing event of PrP C , named γ-cleavage, occurs at the C terminus of PrP C , supposedly between amino acids 176 and 200 (47,48). ...
The prion protein (PrPC) is subjected to several conserved endoproteolytic events producing bioactive fragments that are of increasing interest for their physiological functions and their implication in the pathogenesis of prion diseases and other neurodegenerative diseases. However, systematic and comprehensive investigations on the full spectrum of PrPC proteoforms have been hampered by the lack of methods able to identify all PrPC-derived proteoforms. Building on previous knowledge of PrPC endoproteolytic processing, we thus developed an optimized western blot assay able to obtain the maximum information about PrPC constitutive processing and the relative abundance of PrPC proteoforms in a complex biological sample. This approach led to the concurrent identification of the whole spectrum of known endoproteolytic-derived PrPC proteoforms in brain homogenates, including C-terminal, N-terminal and, most importantly, shed PrPC-derived fragments. Endoproteolytic processing of PrPC was remarkably similar in the brain of widely used wild type and transgenic rodent models, with α-cleavage-derived C1 representing the most abundant proteoform and ADAM10-mediated shedding being an unexpectedly prominent proteolytic event. Interestingly, the relative amount of shed PrPC was higher in wt mice than in most other models. Our results indicate that constitutive endoproteolytic processing of PrPC is not affected by PrPC overexpression or host factors other than PrPC, but can be impacted by PrPC primary structure. Finally, this method represents a crucial step in gaining insight into pathophysiological roles, biomarker suitability, and therapeutic potential of shed PrPC and for a comprehensive appraisal of PrPC proteoforms in therapies, drug screening or in the progression of neurodegenerative diseases.
... 14,38 Similar to SplAsH probes, but different in action, is the AF568-FlAsH probe introduced by Taguchi et al. (Fig. 4). 39 It contains a fully functional FlAsH handle (6-carboxy-FlAsH, isomer of CrAsH) capable of selective conjugation to TC-tagged protein and Alexa Fluor 568. The fluorogenic dyes are conjugated via a cadaveryl linker. ...
... Although this probe has not been deeply characterized, it has been successfully applied for the labelling of extracellular and cell surface proteins, named ''instant with DTT, EDT, and low temperature'' (IDEAL labelling). 39 Analogous to the AF568-FlAsH probe, although different in the structure and optical properties, is AsCy3Cy5, which was obtained by the coupling of unsymmetrical AsCy3 (biarsenical handle) and Cy5 dye (Fig. 4). ...
Fluorescent modification of proteins of interest (POI) in living cells is desired to study their behaviour and functions in their natural environment. In a perfect setting it should be easy to perform, inexpensive, efficient and site-selective. Although multiple chemical and biological methods have been developed, only a few of them are applicable for cellular studies thanks to their appropriate physical, chemical and biological characteristics. One such successful system is a tetracysteine tag/motif and its selective biarsenical binders (e.g. FlAsH and ReAsH). Since its discovery in 1998 by Tsien and co-workers, this method has been enhanced and revolutionized in terms of its efficiency, formed complex stability and breadth of application. Here, we overview the whole field of knowledge, while placing most emphasis on recent reports. We showcase the improvements of classical biarsenical probes with various optical properties as well as multifunctional molecules that add new characteristics to proteins. We also present the evolution of affinity tags and motifs of biarsenical probes demonstrating much more possibilities in cellular applications. We summarize protocols and reported observations so both beginners and advanced users of biarsenical probes can troubleshoot their experiments. We address the concerns regarding the safety of biarsenical probe application. We showcase examples in virology, studies on receptors or amyloid aggregation, where application of biarsenical probes allowed observations that previously were not possible. We provide a summary of current applications ranging from bioanalytical sciences to allosteric control of selected proteins. Finally, we present an outlook to encourage more researchers to use these magnificent probes.
... Furthermore, the occurrence of γ -cleavage has also been suggested recently but at lower frequencies. It was observed in human and animal brain tissue and the neuroblastoma cell line [Taguchi et al., 2009;Lewis et al., 2016]. Considering the molecular mass of the two membrane-bound C-terminal fragments (approximately 18 and 20 kDa after deglycosylation) found in our CAD 5 cells, we assume that they correspond to fragments resulting from αand β-cleavages. ...
Background information:
Cellular prion protein (PrPC ) is infamous for its role in prion diseases. The physiological function of PrPC remains enigmatic, but several studies point to its involvement in cell differentiation processes. To test this possibility, we monitored PrPC changes during the differentiation of prion-susceptible CAD 5 cells, and then we analysed the effect of PrPC ablation on the differentiation process.
Results:
Neuronal CAD 5 cells differentiate within 5 days of serum withdrawal, with the majority of the cells developing long neurites. This process is accompanied by an up to sixfold increase in PrPC expression and enhanced N-terminal β-cleavage of the protein, which suggests a role for the PrPC in the differentiation process. Moreover, the majority of PrPC in differentiated cells is inside the cell, and a large proportion of the protein does not associate with membrane lipid rafts. In contrast, PrPC in proliferating cells is found mostly on the cytoplasmic membrane and is predominantly associated with lipid rafts. To determine the importance of PrPC in cell differentiation, a CAD 5 PrP-/- cell line with ablated PrPC expression was created using the CRISPR/Cas9 system. We observed no considerable difference in morphology, proliferation rate or expression of molecular markers between CAD 5 and CAD 5 PrP-/- cells during the differentiation initiated by serum withdrawal.
Conclusions:
PrPC characteristics, such as cell localisation, level of expression and posttranslational modifications, change during CAD 5 cell differentiation, but PrPC ablation does not change the course of the differentiation process.
Significance:
Ablation of PrPC expression does not affect CAD 5 cell differentiation, although we observed many intriguing changes in PrPC features during the process. Our study does not support the concept that PrPC is important for neuronal cell differentiation, at least in simple in vitro conditions.
... These findings highlighted the potential for the development of a screening assay that focuses on misfolding/aggregating proteins by using a minimal protein tag that should not interfere significantly with the overall folding of the target MisP and the cell-permeable dye FlAsH-EDT2. Tetracysteine motif-binding dyes have also been utilized in order to monitor the misfolding and aggregation of ND-associated MisPs, such as prions, in mammalian cell cultures but not in microbial systems yet [97][98][99][100]. ...
Neurodegenerative diseases (ND) are a major threat to the aging population and the lack of a single preventive or disease-modifying agent only serves to increase their impact. In the past few years, protein misfolding and the subsequent formation of neurotoxic oligomeric/aggregated protein species have emerged as a unifying theme underlying the pathology of these complex diseases. Recently developed microbial genetic screens and selection systems for monitoring ND-associated protein misfolding have allowed the establishment of high-throughput assays for the identification of cellular factors and processes that are important mediators of ND-associated proteotoxicities. In addition, such systems have facilitated the discovery of synthetic and natural compounds with the ability to rescue the misfolding and the associated pathogenic effects of aggregation-prone proteins associated with NDs. This review outlines such available systems in bacteria and yeast, whose usage will likely accelerate the pre-clinical discovery process for effective drugs against a variety of NDs with high socioeconomic impact.
... The exact cleavage site remains to be identified, but molecular weights of the resulting fragments, i.e. a released N-terminal fragments (N3) of~20 kDa and a small GPI-anchored C3 fragment of~5 kDa, indicate cleavage in a region between amino acids 170 and 200, possibly just N-terminal of the first N-glycosylation site (Asn181 in the human and Asn180 in the murine sequence) [34,103] (Fig. 1). Of note, one previous study had described a fragment similar to C3 [104]. Interestingly, γ-cleavage seems to exclusively occur on unglycosylated PrP C indicating that glycans lead to sterical hindrance of the responsible protease, possibly a member of the matrix metalloproteases (MMP) [34,105]. ...
Proteolytic processing of the cellular and disease-associated form of the prion protein leads to generation of bioactive soluble prion protein fragments and modifies the structure and function of its cell-bound form. The nature of proteases responsible for shedding, α-, β-, and γ-cleavage of the prion protein are only partially identified and their regulation is largely unknown. Here, we provide an overview of the increasingly multifaceted picture of prion protein proteolysis and shed light on physiological and pathological roles associated with these cleavages. This article is part of a Special Issue entitled: Proteolysis as a Regulatory Event in Pathophysiology edited by Stefan Rose-John.
... The addition of epitope tags into proteins is a commonly utilized and relatively simple and effective way to study their cellular biology, exploiting the convenience of high affinity antibodies, or intrinsic fluorescence of the tag. The introduction of epitope tags into PrP C which is then expressed in cells or animal tissues has been achieved several times, with different tags, and into different regions of the protein [47][48][49][50][51]. The nucleotide sequence encoding the 10 residue human c-myc epitope (EQKLISEEDL), along with the sequence encoding PrP C residues 226-230 duplicated immediately after the myc-tag, was engineered into murine PRNP immediately 5 0 to the GPI-anchor signal sequence, generating 'PrP-myc'. ...
... A single previous report does identify and describe a small C-terminal PrP fragment of similar apparent molecular mass, which the authors also referred to as 'C3' and may be the same C3 described herein [50]. Taguchi and colleagues determined their fragment to result from cysteine protease cleavage, as a 72 h treatment with a pancysteine protease inhibitor E64 inhibited its production. ...
... A single report does identify and describe a PrP Cderived C-terminal fragment which we originally hypothesized may be the same C3 described herein [50]. The fragment described by Taguchi and colleagues was discovered during the development of a biarsenical cell surface protein labeling technique, using PrP as the model protein. ...
The cellular prion protein (PrP(C)) is a ubiquitously expressed protein of currently unresolved but potentially diverse function. Of putative relevance to normal biological activity, PrP(C) is recognized to undergo both α- and β-endoproteolysis, producing the cleavage fragment pairs N1/C1 and N2/C2, respectively. Experimental evidence suggests the likelihood that these processing events serve differing cellular needs. Through the engineering of a C-terminal c-myc tag onto murine PrP(C), as well as the selective use of a far-C-terminal anti-PrP antibody, we have identified a new PrP(C) fragment, nominally 'C3', and elaborating existing nomenclature, 'γ-cleavage' as the responsible proteolysis. Our studies indicate that this novel γ-cleavage event can occur during transit through the secretory pathway after exiting the endoplasmic reticulum, and after PrP(C) has reached the cell surface, by a matrix metalloprotease. We found that C3 is GPI-anchored like other C-terminal and full length PrP(C) species, though it does not localize primarily at the cell surface, and is preferentially cleaved from an unglycosylated substrate. Importantly, we observed that C3 exists in diverse cell types as well as mouse and human brain tissue, and of possible pathogenic significance, γ-cleavage may increase in human prion diseases. Given the likely relevance of PrP(C) processing to both its normal function, and susceptibility to prion disease, the potential importance of this previously underappreciated and overlooked cleavage event warrants further consideration.
... In order to determine if lysosomal transport of APP is influenced by cell surface antibody binding, we surface labeled HA-βAPP-CFP with a biarsenical-fluorescein reagent (FlAsH), which binds to the engineered Nterminal tetracysteine FlAsH tag in the construct. This labeling technique has been previously used to selectively label cell surface proteins [47] (Fig. 6a). In the absence of antibody, there was robust co-localization of surface labeled APP with Rab5-labeled early endosomes (43.7 ± 3.7 %) at 15 min, whereas only 7.6 ± 2.1 % of this fluorescence was co-localized with LAMP1. ...
... Methods were adapted from Taguchi et al. [47]. Labeling media consisted of 1.4 uM FlAsH reagent (sold under the trade name "Lumio" by Invitrogen), 1 mM 1,2-ethanedithiol (EDT) (Sigma) and 20 mM DL-Dithiothreitol (DTT) (Sigma) incubated together in the dark at room temperature in HBSS for 10 min. ...
Alzheimer’s disease (AD) is characterized by the deposition of Beta-Amyloid (Aβ) peptides in the brain. Aβ peptides are generated by cleavage of the Amyloid Precursor Protein (APP) by the β − and γ − secretase enzymes. Although this process is tightly linked to the internalization of cell surface APP, the compartments responsible are not well defined. We have found that APP can be rapidly internalized from the cell surface to lysosomes, bypassing early and late endosomes. Here we show by confocal microscopy and electron microscopy that this pathway is mediated by macropinocytosis. APP internalization is enhanced by antibody binding/crosslinking of APP suggesting that APP may function as a receptor. Furthermore, a dominant negative mutant of Arf6 blocks direct transport of APP to lysosomes, but does not affect classical endocytosis to endosomes. Arf6 expression increases through the hippocampus with the development of Alzheimer’s disease, being expressed mostly in the CA1 and CA2 regions in normal individuals but spreading through the CA3 and CA4 regions in individuals with pathologically diagnosed AD. Disruption of lysosomal transport of APP reduces both Aβ40 and Aβ42 production by more than 30 %. Our findings suggest that the lysosome is an important site for Aβ production and that altering APP trafficking represents a viable strategy to reduce Aβ production.
Electronic supplementary material
The online version of this article (doi:10.1186/s13041-015-0129-7) contains supplementary material, which is available to authorized users.
... The tags are shown to be preferentially clustered at the N-or C-terminal ends of the sequence, followed by CC2 and the region around α1. N-SS: N-terminal signal sequence; CC1: charge cluster 1; OPR: octapeptide repeat region; CC2: charge cluster 2; PTM: Putative transmembrane region; α1: helix 1; β1: strand 1; α2: helix 2; α3: helix 3; SS-C: C-terminal signal sequence. Types of fusion proteins tested by various groups involved the use of: FLAG 203, 208 , GFP 207-214 , His 204, 215-218 , TC (tetracysteine)196,197,219,220 , AP (alkaline phosphatase)221 , 3F4 222, 223 , L42 224 , Myc 100, 195, 225 and V5 226 epitopes. Shown above the tags are the positions on the protein at which antibodies ICSM35 (CC2) and ICSM18 (α-helix 1), used in this study, bind moPrP. ...
The aim of this study was to systematically investigate the contributions of various amino acids within the prion protein, on prion propagation. To test this in a cellular system, we used a sub-cloned population of N2a cells (PK1) that are highly susceptible to RML mouse prions. A library of stable PK1 cells was generated, which expressed the full length mouse prion protein (moPrP) bearing either point, double, or triple alanine replacements. The effects these changes in the prion protein sequence had on the ability of PK1 cells to propagate RML was tested using a previously established cell based assay. We found that: (i) in the unstructured region of the protein, alanine replacements in CC2 region 90-111 of the prion protein severely diminish, but do not abrogate the ability of cells to propagate prions whilst substitutions K23A.K24A.R25A and Q41A exerted a moderate inhibitory effect on propagation; (ii) alanine replacements in CC2 displayed a dominant negative effect by imposing their propagation inhibition phenotype in the presence of the wild-type protein; (iii) the diminished propagation abilities of cells expressing CC2 alanine mutants were a result of these cells being less susceptible to infection than their wild-type counterparts (iv) all alanine replacements tested in the structured region of the protein appeared to hamper prion propagation, regardless of their positioning within this globular domain. Taken together, these results suggest that integrity of the structured region is vital for successful prion propagation, and that although the flexible region of the prion protein alone (residues 23-111), does not exclusively confer infectivity and/or propagative capacity, charge interactions in this region govern the efficacy with which propagation ensues.
... W metodzie tej jedno z białek zawiera motyw TC, do którego wiąże się selektywnie związek biarsenowy, którego aktywacja promieniowaniem UV prowadzi do kowalencyjnego związania się z drugim białkiem partnerskim [18]. Połączenie barwnika biarsenowego z cząsteczką biotyny pozwala na selektywną heterodimeryzację białek posiadających metkę TC oraz przykładowo streptawidynę, selektywnie wiążącą biotynę [19]. Prosta aktywacja grupy aminowej lub karboksylowej barwnika biarsenowego i związanie go do podłoża stałego, takiego jak żywica czy dwutlenek krzemu, pozwala na wykorzystanie tej klasy związków w selektywnym oczyszczaniu białek na tymże podłożu. ...
Chemical modifications of proteins are crucial for studying their functions, biophysical properties and cellular localization. The most important is the protein fluorescent modification utilized in biochemistry, molecular biology and medicinal diagnostics. Precision of fluorophore attachment in certain part of the protein or amino acid sequence is very important for the labeling and subsequent characterization or protein applications. One of the most important type of probes used for fluorescent protein labeling are biarsenical probes. They are not only to localize proteins in cell but based on their chemical properties they are widely applied for studying protein folding, degradation, control of the activity, protein inactivation with singlet oxygen, oligomerization and protein purification. Here, author presents principles of protein labeling with biarsenical probes with special attention to factors affecting proper protein modification. Review includes the most interesting applications of biarsenical probes in molecular biology, molecular diagnostics and analytical biochemistry.
... Samples were then subjected to gold enhancement per the manufacturer's recommendations (Goldenhance-EM, Nanoprobes). The SEM samples were processed as described elsewhere (61). The TEM samples were processed in a Lynx tissue processor (Electron Microscopy Supplies) as described elsewhere (27). ...
In prion-infected hosts, PrPSc usually accumulates as non-fibrillar, membrane-bound aggregates. Glycosylphosphatidylinositol
(GPI) anchor-directed membrane association appears to be an important factor controlling the biophysical properties of PrPSc
aggregates. To determine whether GPI anchoring can similarly modulate the assembly of other amyloid-forming proteins, neuronal
cell lines were generated that expressed a GPI-anchored form of a model amyloidogenic protein, the NM domain of the yeast
prion protein Sup35 (Sup35GPI). We recently reported that GPI anchoring facilitated the induction of Sup35GPI prions in this system. Here, we report the ultrastructural characterization of self-propagating Sup35GPI aggregates of either spontaneous or induced origin. Like membrane-bound PrPSc, Sup35GPI aggregates resisted release from cells treated with phosphatidylinositol-specific phospholipase C. Sup35GPI aggregates of spontaneous origin were detergent-insoluble, protease-resistant, and self-propagating, in a manner similar
to that reported for recombinant Sup35NM amyloid fibrils and induced Sup35GPI aggregates. However, GPI-anchored Sup35 aggregates were not stained with amyloid-binding dyes, such as Thioflavin T. This
was consistent with ultrastructural analyses, which showed that the aggregates corresponded to dense cell surface accumulations
of membrane vesicle-like structures and were not fibrillar. Together, these results showed that GPI anchoring directs the
assembly of Sup35NM into non-fibrillar, membrane-bound aggregates that resemble PrPSc, raising the possibility that GPI anchor-dependent
modulation of protein aggregation might occur with other amyloidogenic proteins. This may contribute to differences in pathogenesis
and pathology between prion diseases, which uniquely involve aggregation of a GPI-anchored protein, versus other protein misfolding diseases.
