Chemical and Biological Folding Contribute to Temperature-Sensitive ΔF508 CFTR Trafficking

Proteostasis (Balch et al. (2008) Science 319: 916) refers to the biology that maintains the proteome in health and disease. Proteostasis is challenged by the most common mutation in cystic fibrosis, ΔF508, a chloride channel (the cystic fibrosis transmembrane conductance regulator (CFTR)) that exhibits a temperature-sensitive phenotype for coupling to the coatomer complex II (COPII) transport machine for exit from the endoplasmic reticulum (ER). Whether rescue of export of ΔF508-CFTR at reduced temperature simply reflects energetic stabilization of the chemical fold defined by its primary sequence, or requires a unique proteostasis environment is unknown. We now show that reduced temperature (30°C) export of ΔF508 does not occur in some cell types despite efficient export of wild-type CFTR. We find ΔF508 export requires a local biological folding environment that is sensitive to heat/stress inducible factors found in some cell types suggesting that the energetic stabilization by reduced temperature is necessary, but not sufficient for export of ΔF508. Thus, the cell may require a proteostasis environment that is in part distinct from the wild-type pathway to restore ΔF508 coupling to COPII. These results are discussed in the context of the energetics of the protein fold and the potential application of small molecules to achieve a proteostasis environment favoring export of a functional form of ΔF508

Functional Amyloid Formation within Mammalian Tissue

Amyloid is a generally insoluble, fibrous cross-β sheet protein aggregate. The process of amyloidogenesis is associated with a variety of neurodegenerative diseases including Alzheimer, Parkinson, and Huntington disease. We report the discovery of an unprecedented functional mammalian amyloid structure generated by the protein Pmel17. This discovery demonstrates that amyloid is a fundamental nonpathological protein fold utilized by organisms from bacteria to humans. We have found that Pmel17 amyloid templates and accelerates the covalent polymerization of reactive small molecules into melanin—a critically important biopolymer that protects against a broad range of cytotoxic insults including UV and oxidative damage. Pmel17 amyloid also appears to play a role in mitigating the toxicity associated with melanin formation by sequestering and minimizing diffusion of highly reactive, toxic melanin precursors out of the melanosome. Intracellular Pmel17 amyloidogenesis is carefully orchestrated by the secretory pathway, utilizing membrane sequestration and proteolytic steps to protect the cell from amyloid and amyloidogenic intermediates that can be toxic. While functional and pathological amyloid share similar structural features, critical differences in packaging and kinetics of assembly enable the usage of Pmel17 amyloid for normal function. The discovery of native Pmel17 amyloid in mammals provides key insight into the molecular basis of both melanin formation and amyloid pathology, and demonstrates that native amyloid (amyloidin) may be an ancient, evolutionarily conserved protein quaternary structure underpinning diverse pathways contributing to normal cell and tissue physiology

A Chaperone Trap Contributes to the Onset of Cystic Fibrosis

Protein folding is the primary role of proteostasis network (PN) where chaperone interactions with client proteins determine the success or failure of the folding reaction in the cell. We now address how the Phe508 deletion in the NBD1 domain of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) protein responsible for cystic fibrosis (CF) impacts the binding of CFTR with cellular chaperones. We applied single ion reaction monitoring mass spectrometry (SRM-MS) to quantitatively characterize the stoichiometry of the heat shock proteins (Hsps) in CFTR folding intermediates in vivo and mapped the sites of interaction of the NBD1 domain of CFTR with Hsp90 in vitro. Unlike folding of WT-CFTR, we now demonstrate the presence of ΔF508-CFTR in a stalled folding intermediate in stoichiometric association with the core Hsps 40, 70 and 90, referred to as a ‘chaperone trap’. Culturing cells at 30 C resulted in correction of ΔF508-CFTR trafficking and function, restoring the sub-stoichiometric association of core Hsps observed for WT-CFTR. These results support the interpretation that ΔF508-CFTR is restricted to a chaperone-bound folding intermediate, a state that may contribute to its loss of trafficking and increased targeting for degradation. We propose that stalled folding intermediates could define a critical proteostasis pathway branch-point(s) responsible for the loss of function in misfolding diseases as observed in CF

Impact of non-ideal analyte behavior on the separation of protein aggregates by asymmetric flow field-flow fractionation

Asymmetric flow field-flow fractionation is a valuable tool for the characterization of protein aggregates in biotechnology owing to its broad size range and unique separation principle. However, in practice asymmetric flow field-flow fractionation is non-trivial to use due to the major deviations from theory and the influence on separation by various factors that are not fully understood. Here we report methods to assess the non-ideal effects that influence asymmetric flow field-flow fractionation separation and for the first time identify experimentally the main factors that impact it. Furthermore, we propose new approaches to minimize such non-ideal behavior, showing that by adjusting the mobile phase composition (pH and ionic strength) the resolution of asymmetric flow field-flow fractionation separation can be drastically improved. Additionally, we propose a best practice method for new proteins

Determination of the Density of Protein Particles Using a Suspended Microchannel Resonator

One of the analytical tools for characterization of subvisible particles, which gained popularity over the last years because of its unique capabilities, is the resonance mass measurement technique. However, a challenge that this technique presents is the need to know the exact density of the measured particles in order to obtain accurate size calculations. The density of proteinaceous subvisible particles has not been measured experimentally yet and to date researchers have been using estimated density values. In this paper, we report for a first-time experimental measurements of the density of protein particles (0.2-5 μm in size) using particles created by stressing three different proteins using four different types of stress conditions. Interestingly, the particle density values that were measured varied between 1.28 and 1.33 g/cm(3) and were lower than previous estimates. Furthermore, it was found that although the density of proteinaceous particles was affected to a very low degree by the stress conditions used to generate them, there is relatively larger difference between particles originating from different classes of proteins (e.g., monoclonal antibody vs. bovine serum albumin)

Comparative Evaluation of Two Methods for Preparative Fractionation of Proteinaceous Subvisible Particles-Differential Centrifugation and FACS

The goal of this study was to compare and evaluate two preparative techniques for fractionation of proteinaceous subvisible particles. This work enables future studies to address the potential biological consequences of proteinaceous subvisible particles in protein therapeutic products. Particles were generated by heat stress and separated by size using differential centrifugation and FACS (Fluorescence-activated cell sorter). Resulting fractions were characterized by size-exclusion chromatography, light obscuration, flow imaging microscopy and resonant mass measurement. Here we report the optimization and comprehensive evaluation of two methods for preparative fractionation of subvisible proteinaceous particles into distinct size fractions in the range between 0.25 and 100 mu m: differential centrifugation and FACS. Using these methods, well-defined size fractions were prepared and characterized in detail. Critical assessment and comparison of the two techniques demonstrated their complementarity and for the first time-their relative advantages and drawbacks. FACS and differential centrifugation are valuable tools to prepare well-defined size-fractions of subvisible proteinaceous particles. Both techniques possess unique and advantageous attributes and will likely find complementary application in future research on the biological consequences of proteinaceous subvisible particles

Selective Oxidation of Methionine and Tryptophan Residues in a Therapeutic IgG1 Molecule

Oxidation of methionine and tryptophan are common degradation pathways for monoclonal antibodies and present major analytical challenges in biotechnology. Generally, protein oxidation is detectable in stability and/or stressed samples (e.g., exposed to hydrogen peroxide, UV light, or metal ions). The induced chemical modifications may impact the biological activity of antibodies and may have biological consequences. However, these effects and the contribution of individual protein modifications are difficult to delineate as different amino acids are often oxidized simultaneously and accompanied by other degradants such as aggregates, especially in forced degradation studies. Here, we report a new method to obtain selective oxidation of methionine or tryptophan by using oxidation reagents combined with large excess of free tryptophan or methionine, correspondingly. More specifically, using hydrogen peroxide or tert-butyl hydroperoxide in combination with addition of free tryptophan allowed for selective oxidation of methionine. Conversely, the use of 2,2-azobis(2-amidinopropane) dihydrochloride in combination with free methionine resulted in selective tryptophan oxidation, whereas methionine oxidation was not significantly altered. This novel stress model system may prove to be valuable tool in future mechanistic studies of oxidative degradation of protein therapeutics

Measuring Subvisible Particles in Protein Formulations Using a Modified Light Obscuration Sensor with Improved Detection Capabilities

Although light obscuration is the "gold standard" for subvisible particle measurements in biopharmaceutical products, the current technology has limitations with respect to the detection of translucent proteinaceous particles and particles of sizes smaller and around 2 μm. Here, we describe the evaluation of a modified light obscuration sensor utilizing a novel measuring mode. Whereas standard light obscuration methodology monitors the height (amplitude) of the signal, the new approach monitors its length (width). Experimental evaluation demonstrated that this new detection mode leads to improved detection of subvisible particles of sizes smaller than 2 μm, reduction of artifacts during measurements especially of low concentrations of translucent protein particles, and higher counting accuracy as compared to flow imaging microscopy and standard light obscuration measurements

Factors Governing the Precision of Subvisible Particle Measurement Methods - A Case Study with a Low-Concentration Therapeutic Protein Product in a Prefilled Syringe

PURPOSE: The current study was performed to assess the precision of the principal subvisible particle measurement methods available today. Special attention was given to identifying the sources of error and the factors governing analytical performance. METHODS: The performance of individual techniques was evaluated using a commercial biologic drug product in a prefilled syringe container. In control experiments, latex spheres were used as standards and instrument calibration suspensions. RESULTS: The results reported in this manuscript clearly demonstrated that the particle measurement techniques operating in the submicrometer range have much lower precision than the micrometer size-range methods. It was established that the main factor governing the relatively poor precision of submicrometer methods in general and inherently, is their low sampling volume and the corresponding large extrapolation factors for calculating final results. CONCLUSIONS: The variety of new methods for submicrometer particle analysis may in the future support product characterization; however, the performance of the existing methods does not yet allow for their use in routine practice and quality control