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Structure and Function of Energy-Dependent Protein-Remodeling Factors

Objective

Project 1: Mechanism of protein disaggregation mediated by ClpB <BR> Objectives: 1.1 Experimentally verify predictions of the aggregation-promoting protein motifs made by computational modeling of the protein aggregation process [yr 1-2]. <BR>1.2 Determine whether the aggregation-promoting motifs are preferentially recognized by ClpB [yr 2-4]. <BR>1.3 Determine which protein motifs are primarily extracted from aggregates by ClpB [yr 2-4]. <BR>1.4 Characterize conformational rearrangements in ClpB that mediate substrate extraction from an aggregate [yr 3-5]. <BR> <BR>Project 2: Biochemical characterization of the torsin sub-family of AAA+ ATPases <BR>Objectives: 2.1 Develop expression systems for production and purification of torsins (besides torsinA, which has been already purified in our laboratory) [yr 1-2] <BR>2.2 Perform a bioinformatic analysis of the AAA+ super-family to identify sequence motifs which possibly co-evolved with the torsin-like non-canonical Walker A [yr 1-2]. <BR>2.3 Compare the biochemical properties of torsins containing the non-canonical Walker A motif with those of the torsin containing the canonical Walker A [yr 2-4]. <BR> 2.4 Test cellular co-localization and possible interaction and/or functional cooperation between different torsins [yr 3-5]. <BR> <BR>The outcome of this Action Plan will be a significant amount of new data, conclusions, and predictions that will be useful for scientists working in different fields of biochemistry and cell biology. We do not anticipate immediate industrial applications of our results. The results of this research will be communicated to the scientific community in the form of conference posters, seminars, and peer-reviewed publications.

More information

NON-TECHNICAL SUMMARY: This is a basic-research Action Plan that will advance our understanding of the biological function and mechanism of selected proteins that play essential roles in physiological processes by remodeling structure and conformation of their target macromolecules. The relevance of such basic biochemical research lies in its potential to fill a significant gap in our knowledge of the molecular physiology of living organisms with possible practical applications in the future. Specifically, one project of the plan will focus on the mechanism of reactivation of aggregated proteins by ClpB, a molecular chaperone from the AAA+ family of ATPases. Protein aggregation is a major impediment in many biotechnological processes and, importantly, it is a culprit of many pathological conditions in humans, animals, and plants. Prion disease (also known as mad-cow disease) is an example of an aggregation-induced lethal animal pathology that can be transmitted to humans. So far, mad-cow disease has mostly affected the beef industry and the agricultural economics in Europe, not in the U.S., but a continuing research on prion aggregation is essential for health of the population and for prosperity of all beef-producing regions, including Kansas. Our second project will focus on torsins, a family of animal AAA+ ATPases linked to several essential physiological processes, such as protein secretion, transmembrane-protein transport, vesicular transport, and nuclear-envelope biogenesis. The precise biological role of torsins is unknown and our studies may contribute to understanding the molecular regulation of the above pathways. In summary, our basic research may benefit human and animal health and may find applications in food production, processing, and quality control.

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APPROACH: <BR>Project 1: Mechanism of protein disaggregation mediated by ClpB <BR>1.1 Verify predictions of the aggregation-promoting protein motifs made by computational modeling of the protein aggregation process. We will use fluorescein isothiocyanate (FITC) to label all exposed groups at the surface of G6PDH at different stages of aggregation. We will then produce tryptic peptides from G6PDH and identify the peptide sequences using mass spectrometry. <BR>1.2 Determine whether the aggregation-promoting motifs are preferentially recognized by ClpB. We will use fluorescence resonance energy transfer (FRET) between donor-labeled ClpB and acceptor-labeled G6PDH to investigate interactions between ClpB and an aggregate. We will compare FRET efficiencies produced by the donor-acceptor pairs located in different sites in ClpB and in different regions of the aggregated substrate. <BR>1.3 Determine which protein motifs are primarily extracted from aggregates by ClpB. We will use a stopped-flow fluorometer to observe the kinetics of FRET development during translocation of substrates labeled at different sites. <BR>1.4 Characterize conformational rearrangements in ClpB that mediate substrate extraction from an aggregate. We will use a fluorescence response of a single Trp engineered to replace a Tyr at the channel entrance. The fluorescence response of Trp will be evaluated for different ClpB mutants with modified mobility of specific structural domains. <BR> <BR>Project 2: Biochemical characterization of the torsin sub-family of AAA+ ATPases <BR>2.1 Develop expression systems for production and purification of torsins. We will obtain DNA constructs of human or mouse torsin1B, torsin2, and torsin3 and sub-clone their coding regions into the yeast and insect expression vectors. We will then establish procedures for purification of torsins. <BR>2.2 Perform a bioinformatic analysis of the AAA+ super-family to identify sequence motifs which possibly co-evolved with the torsinA-like non-canonical Walker A. We will develop procedures for the analysis of multiple AAA+ sequences to find statistically-significant correlations between Asn in Walker A and other sequence variations. <BR>2.3 Compare the biochemical properties of torsins containing the non-canonical Walker A motif with those of torsin2 containing the canonical Walker A. We will produce mutant torsins with their Walker A sequence converted to the canonical one and test which biochemical properties are directly controlled by that motif. <BR>2.4 Test cellular co-localization and possible interaction and/or functional cooperation between different torsins. <BR> <BR>We will establish mammalian cell lines with overproduction of each torsin. We will determine the sub-cellular localization of torsins. We will investigate whether mutations of the Walker A sequence or other identified co-evolving sequences affect the localization. We will perform immunoprecipitation of torsins and test whether other family members co-immunoprecipitate. We will use purified torsins to perform pull-down assays and search for interacting partners of torsins using proteomic approaches.

Institution
Kansas State University
Start date
2008
End date
2013
Project number
KS405
Accession number
213749
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