19 research outputs found
Quantitative Proteomic Analysis of Stratum Corneum Dysfunction in Adult Chronic Atopic Dermatitis.
Application of AllerCatPro 2.0 for protein safety assessments of consumer products
Foreign proteins are potentially immunogenic, and a proportion of these are able to induce immune responses that result in allergic sensitization. Subsequent exposure of sensitized subjects to the inducing protein can provoke a variety of allergic reactions that may be severe, or even fatal. It has therefore been recognized for some time that it is important to determine a priori whether a given protein has the potential to induce allergic responses in exposed subjects. For example, the need to assess whether transgene products expressed in genetically engineered crop plants have allergenic properties. This is not necessarily a straightforward exercise (as discussed elsewhere in this edition), but the task becomes even more challenging when there is a need to conduct an overall allergenicity safety assessment of complex mixtures of proteins in botanicals or other natural sources that are to be used in consumer products. This paper describes a new paradigm for the allergenicity safety assessment of proteins that is based on the use of AllerCatPro 2.0, a new version of a previously described web application model developed for the characterization of the allergenic potential of proteins. Operational aspects of AllerCatPro 2.0 are described with emphasis on the application of new features that provide improvements in the predictions of allergenic properties such as the identification of proteins with high allergenic concern. Furthermore, the paper provides a description of strategies of how AllerCatPro 2.0 can best be deployed as a screening tool for identifying suitable proteins as ingredients in consumer products as well as a tool, in conjunction with label-free proteomic analysis, for identifying and semiquantifying protein allergens in complex materials. Lastly, the paper discusses the steps that are recommended for formal allergenicity safety assessment of novel consumer products which contain proteins, including consideration and integration of predicted consumer exposure metrics. The article therefore provides a holistic perspective of the processes through which effective protein safety assessments can be made of potential allergenic hazards and risks associated with exposure to proteins in consumer products, with a particular focus on the use of AllerCatPro 2.0 for this purpose
Niclosamide Prevents the Formation of Large Ubiquitin-Containing Aggregates Caused by Proteasome Inhibition
Protein aggregation is a hallmark of many neurodegenerative diseases and has been linked to the failure to degrade misfolded and damaged proteins. In the cell, aberrant proteins are degraded by the ubiquitin proteasome system that mainly targets short-lived proteins, or by the lysosomes that mostly clear long-lived and poorly soluble proteins. Both systems are interconnected and, in some instances, autophagy can redirect proteasome substrates to the lysosomes.To better understand the interplay between these two systems, we established a neuroblastoma cell population stably expressing the GFP-ubiquitin fusion protein. We show that inhibition of the proteasome leads to the formation of large ubiquitin-containing inclusions accompanied by lower solubility of the ubiquitin conjugates. Strikingly, the formation of the ubiquitin-containing aggregates does not require ectopic expression of disease-specific proteins. Moreover, formation of these focused inclusions caused by proteasome inhibition requires the lysine 63 (K63) of ubiquitin. We then assessed selected compounds that stimulate autophagy and found that the antihelmintic chemical niclosamide prevents large aggregate formation induced by proteasome inhibition, while the prototypical mTORC1 inhibitor rapamycin had no apparent effect. Niclosamide also precludes the accumulation of poly-ubiquitinated proteins and of p62 upon proteasome inhibition. Moreover, niclosamide induces a change in lysosome distribution in the cell that, in the absence of proteasome activity, may favor the uptake into lysosomes of ubiquitinated proteins before they form large aggregates.Our results indicate that proteasome inhibition provokes the formation of large ubiquitin containing aggregates in tissue culture cells, even in the absence of disease specific proteins. Furthermore our study suggests that the autophagy-inducing compound niclosamide may promote the selective clearance of ubiquitinated proteins in the absence of proteasome activity
In vitro investigations into the structures of membrane-associated proteins augmented by the application of computational methods
Approximately half of all proteins are associated with ordered lipid aggregates such as the plasma membrane. These molecules are involved in an array of processes critical for the maintenance of life, including signaling, environmental sensing, import/export, homeostasis, and cellular structure. Despite the crucial role that membrane-associated proteins play, we know less about them than we do of their soluble counterparts. This is due primarily to technical limitations of the standard techniques employed for producing and studying biological macromolecules, which often work best for samples in which the protein is soluble in aqueous buffer at high concentrations. Due to their ability to interact with hydrophobic lipids, membrane-associated proteins frequently pose complications to the researcher in this sample production phase. Recent advances in techniques and scientific methods (driven by a strong desire to elucidate the workings of the membrane systems) have led to a rapid expansion in our knowledge; both the basic information needed to effectively work with these types of molecules as well as an increase in our biochemical dataset of their functions. This is not to say that obtaining data on membrane-associated proteins is now facile. It is critical to leverage the appropriate tools in order to extract the maximum amount of information from the system being studied. As such, this document describes the course of my own work into several membrane-associated proteins. In all of these studies I employed computational methods such as molecular modeling to guide the work. The in silico predictions were useful in increasing protein production, allowed for comparisons across a family of similar proteins, and suggested experimental lines of inquiry. At the same time, disagreements between simulations and in vitro data reinforced the necessity of obtaining experimental results. The first three chapters of this dissertaion detail my work with G Protein-Coupled Receptors (GPCRs). These integral, polytopic transmembrane polypeptides are one of the largest superfamilies of proteins and are central players in signal transduction across a lipid bilayer. I describe the design and initial generation steps of a ligand binding analog of the Tachykinin NK1 Receptor which is more amenable to structural analysis. In this case a homology model of NK1 was used during the analog design phase. Distance information from this model was applied to determine the lengths of linker sequences to include between ligand binding segments of the receptor. The partial genes for these segments were then synthesised and combined to create the full analog gene. We then expressed, purified, and characterized the protein. I also describe my investigations into the activation of the unfolded protein response in Saccharomyces cerevisiae when the NK1 receptor is heterologously expressed. We found that overexpression of NK1 in yeast induced the Unfolded Protein Response, in contrast to the Adenosine A2A receptor which did not. The next four chapters deal with my experiments involving a family of peripheral membrane associated proteins, the Phospholipases A2 (PLA2). I describe the generation of a homology model of the Platelet Activating Factor Acetyl Hydrolase Type 2 (PAFAH2) based on a template structure recently solved in our lab. This model was used to help guide our mutagenesis experiments to further understand the activation of the protein under conditions of oxidative stress, as well as to draw comparisons with the structure of the related plasma PAFAH. I then move on to cover work on the small secreted PLA2 family. This project primarily focused on elucidation of the causes of inhibitor specificity between the human group V and group X proteins. In order to facilitate model building we developed software tools which combined disulfide bond definition, heteroatom inclusion, structurally influenced alignments, and scoring of the produced models. For both of the PLA2 projects we found that these methods were necessary in order to generate acceptable homology models, and were superior to “black-box” automated algorithms. For the sPLA 2s, the homology model of the human Group V protein allowed us to compare the features of this protein with other physiologically relevant family members in order to connect structural features with biological function. Once again, the common thread throughout the work described in this dissertation is the augmentation of in vitro experiments with in silico calculations. This application of predictive tools acts as a means of maximizing research time and optimizing the use of the limited material produced in the laboratory