School of Biomedical Sciences - Research Publications
Permanent URI for this collection
Search Results
1 - 6 of 6
-
ItemDiagnostics for Amyloid Fibril Formation: Where to Begin?(HUMANA PRESS INC, 2011)Twenty-five proteins are known to form amyloid fibrils in vivo in association with disease (Westermark et al., Amyloid 12:1-4, 2005). However, the fundamental ability of a protein to form amyloid-like fibrils is far more widespread than in just the proteins associated with disease, and indeed this property can provide insight into the basic thermodynamics of folding and misfolding pathways. But how does one determine whether a protein has formed amyloid-like fibrils? In this chapter, we cover the basic steps toward defining the amyloid-like properties of a protein and how to measure the kinetics of fibrillization. We describe several basic tests for aggregation and the binding to two classic amyloid-reactive dyes, Congo Red, and thioflavin T, which are key indicators to the presence of fibrils.
-
ItemIdentifying polyglutamine protein species in situ that best predict neurodegeneration(NATURE PUBLISHING GROUP, 2011-12)Polyglutamine (polyQ) stretches exceeding a threshold length confer a toxic function to proteins that contain them and cause at least nine neurological disorders. The basis for this toxicity threshold is unclear. Although polyQ expansions render proteins prone to aggregate into inclusion bodies, this may be a neuronal coping response to more toxic forms of polyQ. The exact structure of these more toxic forms is unknown. Here we show that the monoclonal antibody 3B5H10 recognizes a species of polyQ protein in situ that strongly predicts neuronal death. The epitope selectively appears among some of the many low-molecular-weight conformational states assumed by expanded polyQ and disappears in higher-molecular-weight aggregated forms, such as inclusion bodies. These results suggest that protein monomers and possibly small oligomers containing expanded polyQ stretches can adopt a conformation that is recognized by 3B5H10 and is toxic or closely related to a toxic species.
-
ItemReAsH/FlAsH Labeling and Image Analysis of Tetracysteine Sensor Proteins in Cells(JOURNAL OF VISUALIZED EXPERIMENTS, 2011-08)Fluorescent proteins and dyes are essential tools for the study of protein trafficking, localization and function in cells. While fluorescent proteins such as green fluorescence protein (GFP) have been extensively used as fusion partners to proteins to track the properties of a protein of interest, recent developments with smaller tags enable new functionalities of proteins to be examined in cells such as conformational change and protein-association. One small tag system involves a tetracysteine motif (CCXXCC) genetically inserted into a target protein, which binds to biarsenical dyes, ReAsH (red fluorescent) and FlAsH (green fluorescent), with high specificity even in live cells. The TC/biarsenical dye system offers far less steric constraints to the host protein than fluorescent proteins which has enabled several new approaches to measure conformational change and protein-protein interactions. We recently developed a novel application of TC tags as sensors of oligomerization in cells expressing mutant huntingtin, which when mutated aggregates in neurons in Huntington disease. Huntingtin was tagged with two fluorescent dyes, one a fluorescent protein to track protein location, and the second a TC tag which only binds biarsenical dyes in monomers. Hence, changes in colocalization between protein and biarsenical dye reactivity enabled submicroscopic oligomer content to be spatially mapped within cells. Here, we describe how to label TC-tagged proteins fused to a fluorescent protein (Cherry, GFP or CFP) with FlAsH or ReAsH in live mammalian cells and how to quantify the two color fluorescence (Cherry/FlAsH, CFP/FlAsH or GFP/ReAsH combinations).
-
ItemPOLYGLUTAMINE AGGREGATION IN HUNTINGTON AND RELATED DISEASES(SPRINGER-VERLAG BERLIN, 2012)Polyglutamine (polyQ)-expansions in different proteins cause nine neurodegenerative diseases. While polyQ aggregation is a key pathological hallmark of these diseases, how aggregation relates to pathogenesis remains contentious. In this chapter, we review what is known about the aggregation process and how cells respond and interact with the polyQ-expanded proteins. We cover detailed biophysical and structural studies to uncover the intrinsic features of polyQ aggregates and concomitant effects in the cellular environment. We also examine the functional consequences ofpolyQ aggregation and how cells may attempt to intervene and guide the aggregation process.
-
ItemTracking protein aggregation and mislocalization in cells with flow cytometry(NATURE PUBLISHING GROUP, 2012-05)We applied pulse-shape analysis (PulSA) to monitor protein localization changes in mammalian cells by flow cytometry. PulSA enabled high-throughput tracking of protein aggregation, translocation from the cytoplasm to the nucleus and trafficking from the plasma membrane to the Golgi as well as stress-granule formation. Combining PulSA with tetracysteine-based oligomer sensors in a cell model of Huntington's disease enabled further separation of cells enriched with monomers, oligomers and inclusion bodies.
-
ItemPutting Huntingtin "Aggregation" in View with Windows into the Cellular Milieu(BENTHAM SCIENCE PUBL LTD, 2012-11)Huntington's disease arises from CAG codon-repeat expansions in the Htt gene, which leads to a Htt gene product with an expanded polyglutamine (polyQ) sequence. The length of the polyQ expansion correlates with an increased tendency to form aggregates and clustering into micrometer-plus sized inclusion bodies in neurons and other cell types. Yet after nearly 20 years since the genetic basis for HD was identified, our knowledge of how polyQ-expanded Htt fragment aggregation relates to disease mechanisms remains fragmentary and controversial. Challenges remain in defining the aggregation process at the molecular level and how this process is influenced by, or influences cellular activities. Insight is further confounded by the term "aggregation" being used to describe a composite of distinct processes that may have opposing consequences to cell health and survival. This review discusses these issues in light of a historic summary of Htt aggregation in the cellular milieu and the intrinsic attributes of polyQ-expanded Htt that lead to aggregation. Finally, discussion centers on strategies forward to improve our knowledge for how aggregation relates to cellular dysfunction.