Karen C. Sitney

BOTH FAMILIAL PARKINSON'S DISEASE MUTATIONS ACCELERATE ALPHA -SYNUCLEIN AGGREGATION

Parkinson’s disease (PD) is a neurodegenerative disorder that is pathologically characterized by the presence of intracytoplasmic Lewy bodies, the major component of which are filaments consisting of α-synuclein. Two recently identified point mutations in α-synuclein are the only known genetic causes of PD, but their pathogenic mechanism is not understood. Here we show that both wild type and mutant α-synuclein form insoluble fibrillar aggregates with antiparallel β-sheet structure upon incubation at physiological temperature in vitro. Importantly, aggregate formation is accelerated by both PD-linked mutations. Under the experimental conditions, the lag time for the formation of precipitable aggregates is about 280 h for the wild type protein, 180 h for the A30P mutant, and only 100 h for the A53T mutant protein. These data suggest that the formation of α-synuclein aggregates could be a critical step in PD pathogenesis, which is accelerated by the PD-linked mutations.

Efficiency of signalling through cytokine receptors depends critically on receptor orientation

Human erythropoietin is a haematopoietic cytokine required for the differentiation and proliferation of precursor cells into red blood cells. It activates cells by binding and orientating two cell-surface erythropoietin receptors (EPORs) which trigger an intracellular phosphorylation cascade. The half-maximal response in a cellular proliferation assay is evoked at an erythropoietin concentration of 10 pM (ref. 3), 10−2 of its K d value for erythropoietin–EPOR binding site 1 (Kd ≈ 1 nM), and 10−5 of the K d for erythropoietin–EPOR binding site 2 (Kd ≈ 1 μM). Overall half-maximal binding (IC50) of cell-surface receptors is produced with ∼0.18 nM erythropoietin, indicating that only ∼6% of the receptors would be bound in the presence of 10 pM erythropoietin. Other effective erythropoietin-mimetic ligands that dimerize receptors can evoke the same cellular responses, but much less efficiently, requiring concentrations close to their K d values (∼0.1 μM). The crystal structure of erythropoietin complexed to the extracellular ligand-binding domains of the erythropoietin receptor, determined at 1.9 Å from two crystal forms, shows that erythropoietin imposes a unique 120° angular relationship and orientation that is responsible for optimal signalling through intracellular kinase pathways.

Interactions between NFκB and Its Inhibitor iκB: Biophysical Characterization of a NFκB/iκB-α Complex

The N-terminal domain (1–318 amino acids) of mouse NFκB (p65) has been purified to homogeneity from the soluble fraction of Escherichia coli cells expressing this protein. Its complex with a full-length iκB-α (MAD3, 1–317 amino acids) molecule was generated by binding the E. coli-derived iκB-α to the purified NFκB and purifying the complex by sequential chromatography. The stoichiometry of NFκB to iκB in the complex was determined to be 2 to 1 by light scattering and SDS–polyacrylamide gel electrophoresis. The secondary structure of the NFκB (p65) determined by Fourier-transform infrared (FTIR) spectroscopy is in good agreement with that of the p50 in the crystal structure of the p50/DNA complex, indicating that no significant structural change in NFκB occurs upon binding of DNA. The FTIR spectrum of the NFκB/iκB complex indicates that its secondary structure is composed of 17% α-helix, 39% β-strand, 18% irregular structures, and 26% β-turns and loops. By comparing these data to the FTIR data for NFκB alone, it is concluded that the iκB (MAD3) in the complex contains 35% α-helix, 27% β-strand, 22% irregular structures, and 16% β-turns and loops. Circular dichroism (CD) analysis of a shorter form of iκB (pp40) indicates that it contains at least 20% α-helix and that the iκB subunit accounts for nearly all of the α-helix present in the NFκB/iκB complex, consistent with the FTIR results. The stabilities of NFκB, iκB, and their complex against heat-induced denaturation were investigated by following changes in CD signal. The results indicate that the thermal stability of iκB is enhanced upon the formation of the NFκB/iκB complex.

Use of a modified tryptophanase promoter to direct high-level expression of foreign proteins in E. coli.

We have modified the tryptophanase promoter (PtnaA) for use as a temperature-independent promoter for the production of recombinant proteins. Although any protein will have a temperature range in which its expression is optimal, we find the tryptophanase promoter functions at all physiologically relevant temperatures (20 degrees C to 42 degrees C). Induction at temperatures below 37 degrees C avoids eliciting the heat-shock response and may favor the production of protein in the soluble state. A short segment of the E. coli tnaA promoter containing the catabolite gene activator protein (CAP) binding site but no tryptophan-responsive elements was used to direct synthesis of various proteins. Conditions for high cell density fermentation and induction control were developed. Expression was induced by depletion of glucose and was maximal when an alternative nonrepressing carbon source was supplied. Expression of certain proteins was tightly controlled; however, pre-induction expression was observed with other reporter genes. The tnaC leader portion of the tnaA promoter was found to reduce pre-induction expression in the presence of glucose, although maximal expression was observed only in the absence of this region. The effect of temperature on expression of several recombinant proteins was investigated. Although some proteins were produced only in inclusion bodies as insoluble material, the production of one protein in soluble form was clearly temperature dependent.

Protein Drug Manufacturing

Recombinant DNA technology has opened new avenues for producing many useful therapeutic proteins, such as hormones, growth factors, and interferons, in commercial quantities. To economically produce therapeutic proteins at the commercial scale, while controlling product quality, requires three general steps. First, an effective strategy for maximizing recombinant gene expression must be devised. Next, an efficient fermentation process must be developed. Finally, robust, effective protein recovery and purification processes must be developed.