Development of a methodology for large-scale production of prions for biological and structural studies

Prion diseases are a group of infectious neurodegenerative diseases produced by the conversion of the normal prion protein (PrPC) into the disease-associated form (PrPSc). Extensive evidence indicate that the main or sole component of the infectious agent is PrPSc, which can replicate in affected individuals in the absence of nucleic acids. However, the mechanism of PrPC-to-PrPSc conversion remains elusive, which has been attributed to the lack of sufficient structural information of infectious PrPSc and a reliable system to study prion replication in vitro. In this article we adapted the Protein Misfolding Cyclic Amplification (PMCA) technology for rapid and efficient generation of highly infectious prions in large-scale. Murine prions of the RML strain were efficiently propagated in volumes up to 1,000-fold larger than conventional PMCA. The large-scale PMCA (LS-PMCA) procedure enabled to produce highly infectious prions, which maintain the strain properties of the seed used to begin the reaction. LS-PMCA was shown to work with various species and strains of prions, including mouse RML and 301C strains, hamster Hyper prion, cervid CWD prions, including a rare Norwegian CWD prion, and human CJD prions. We further improved the LS-PMCA into a bioreactor format that can operate under industry-mimicking conditions for continuous and unlimited production of PrPSc without the need to keep adding brain-derived prions. In our estimation, this bioreactor can produce in 1d an amount of prions equivalent to that present in 25 infected animals at the terminal stage of the disease. Our LS-PMCA technology may provide a valuable tool to produce large quantities of well-defined and homogeneous infectious prions for biological and structural studies

Detection of prions in blood from patients with variant Creutzfeldt-Jakob disease

Prions can be detected in blood from patients with variant Creutzfeldt-Jakob disease with high sensitivity and specificity.</jats:p

Prions in the Urine of Patients with Variant Creutzfeldt–Jakob Disease

BACKGROUND: Prions, the infectious agents responsible for transmissible spongiform encephalopathies, consist mainly of the misfolded prion protein (PrP(Sc)). The unique mechanism of transmission and the appearance of a variant form of Creutzfeldt–Jakob disease, which has been linked to consumption of prion-contaminated cattle meat, have raised concerns about public health. Evidence suggests that variant Creutzfeldt–Jakob disease prions circulate in body fluids from people in whom the disease is silently incubating. METHODS: To investigate whether PrP(Sc) can be detected in the urine of patients with variant Creutzfeldt–Jakob disease, we used the protein misfolding cyclic amplification (PMCA) technique to amplify minute quantities of PrP(Sc), enabling highly sensitive detection of the protein. We analyzed urine samples from several patients with various transmissible spongiform encephalopathies (variant and sporadic Creutzfeldt–Jakob disease and genetic forms of prion disease), patients with other degenerative or nondegenerative neurologic disorders, and healthy persons. RESULTS: PrP(Sc) was detectable only in the urine of patients with variant Creutzfeldt–Jakob disease and had the typical electrophoretic profile associated with this disease. PrP(Sc) was detected in 13 of 14 urine samples obtained from patients with variant Creutzfeldt–Jakob disease and in none of the 224 urine samples obtained from patients with other neurologic diseases and from healthy controls, resulting in an estimated sensitivity of 92.9% (95% confidence interval [CI], 66.1 to 99.8) and a specificity of 100.0% (95% CI, 98.4 to 100.0). The PrP(Sc) concentration in urine calculated by means of quantitative PMCA was estimated at 1×10(−16) g per milliliter, or 3×10(−21) mol per milliliter, which extrapolates to approximately 40 to 100 oligomeric particles of PrP(Sc) per milliliter of urine. CONCLUSIONS: Urine samples obtained from patients with variant Creutzfeldt–Jakob disease contained minute quantities of PrP(Sc). (Funded by the National Institutes of Health and others.

Thermal adaptation of mesophilic and thermophilic FtsZ assembly by modulation of the critical concentration

Cytokinesis is the last stage in the cell cycle. In prokaryotes, the protein FtsZ guides cell constriction by assembling into a contractile ring-shaped structure termed the Z-ring. Constriction of the Z-ring is driven by the GTPase activity of FtsZ that overcomes the energetic barrier between two protein conformations having different propensities to assemble into polymers. FtsZ is found in psychrophilic, mesophilic and thermophilic organisms thereby functioning at temperatures ranging from subzero to >100 degrees C. To gain insight into the functional adaptations enabling assembly of FtsZ in distinct environmental conditions, we analyzed the energetics of FtsZ function from mesophilic Escherichia coli in comparison with FtsZ from thermophilic Methanocaldococcus jannaschii. Presumably, the assembly may be similarly modulated by temperature for both FtsZ orthologs. The temperature dependence of the first-order rates of nucleotide hydrolysis and of polymer disassembly, indicated an entropy-driven destabilization of the FtsZ-GTP intermediate. This destabilization was true for both mesophilic and thermophilic FtsZ, reflecting a conserved mechanism of disassembly. From the temperature dependence of the critical concentrations for polymerization, we detected a change of opposite sign in the heat capacity, that was partially explained by the specific changes in the solvent-accessible surface area between the free and polymerized states of FtsZ. At the physiological temperature, the assembly of both FtsZ orthologs was found to be driven by a small positive entropy. In contrast, the assembly occurred with a negative enthalpy for mesophilic FtsZ and with a positive enthalpy for thermophilic FtsZ. Notably, the assembly of both FtsZ orthologs is characterized by a critical concentration of similar value (1-2 mu M) at the environmental temperatures of their host organisms. These findings suggest a simple but robust mechanism of adaptation of FtsZ, previously shown for eukaryotic tubulin, by adjustment of the critical concentration for polymerization.Becas Chile and Programa de Mejoramiento de la Calidad y Equidad de la Educacion Comision Nacional de Investigacion Cientifica y Tecnologica 24090139 Fondo Nacional de Desarrollo Cientifico y Tecnologico 113071

Eyring plots of the GTPase activity and polymerization of FtsZ.

<p>Top row, mesophilic EcFtsZ. Bottom row, thermophilic MjFtsZ. The kinetic rates of GTP hydrolysis, <i>k_cat</i> (A and C), and filament depolymerization, <i>k_depol</i> (B and D), are plotted as a function of temperature. The apparent enthalpies (Δ<i>H</i>^0‡) and entropies (Δ<i>S</i>^0‡) of the transition state were calculated by nonlinear regression using <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185707#pone.0185707.e006" target="_blank">Eq 4</a> (solid lines), and the best-fit values are reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185707#pone.0185707.t003" target="_blank">Table 3</a>.</p

The morphology of FtsZ polymers was characterized by negative-stain transmission electron microscopy.

<p>Top row, 10 μM EcFtsZ was polymerized at 10, 20 and 30°C (A-C). Bottom row, 10 μM MjFtsZ was polymerized at 40, 60 and 80°C (D–F). The arrowheads in D point to examples of short and curved polymers. The scale bars represent 100 and 500 nm for the top and bottom rows, respectively (black bars).</p

Global analysis of the critical concentration using the integrated van′t Hoff equation.

<p>The combined data obtained with the GTP hydrolysis and polymerization assays were fit to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185707#pone.0185707.e008" target="_blank">Eq 6</a> using nonlinear regression (solid lines), for mesophilic EcFtsZ (A) and thermophilic MjFtsZ (B). The fitting coefficients for EcFtsZ are: <i>a</i> = -787.59, <i>b</i> = 38,745 and <i>c</i> = 117.86, and the coefficients obtained for MjFtsZ: <i>a</i> = 776.24, <i>b</i> = -40,253 and <i>c</i> = -110.53. The temperature-dependent parameters Δ<i>H</i>⁰ and Δ<i>S</i>⁰ were calculated using <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185707#pone.0185707.e010" target="_blank">Eq 7</a> (Panels C and D), as well as the temperature-independent heat capacity change Δ<i>C</i>_<i>p</i> (see text).</p

Measured parameters for the GTPase activity and polymerization of FtsZ from <i>Methanocaldococcus jannaschii</i>.

<p>Measured parameters for the GTPase activity and polymerization of FtsZ from <i>Methanocaldococcus jannaschii</i>.</p

Measured parameters for the GTPase activity and polymerization of FtsZ from <i>Escherichia coli</i>.

<p>Measured parameters for the GTPase activity and polymerization of FtsZ from <i>Escherichia coli</i>.</p

Temperature dependence of the GTP hydrolysis rates of FtsZ.

<p>The effect of temperature and protein concentration on the GTPase activity of FtsZ was examined for mesophilic EcFtsZ (A) and for thermophilic MjFtsZ (B). The catalytic constant <i>k_cat</i> was determined from the slopes of the linear regressions indicated by the solid lines (the substrate was used at saturating concentrations). The critical concentrations C_C-GTPase were obtained from the intersection of the linear regressions with the protein concentration axis. The symbols and error bars are the averages and the standard deviations from triplicate samples. The calculated values of C_C-GTPase and <i>k_cat</i> are presented in Tables <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185707#pone.0185707.t001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185707#pone.0185707.t002" target="_blank">2</a>.</p