The long-term goal of the Miller lab is to understand how signal transduction circuits control microtubules so that effective and targeted interventions can be developed to manipulate microtubule functions in stressful situations like disease and space travel. The Miller lab recently established that the microtubule-associated protein Stu2p interacts with SUMO. Preliminary data from the Miller lab suggest that there are two modes of interaction between Stu2p and SUMO: a covalent interaction in which Stu2p is conjugated by SUMO and a non-covalent interaction in which Stu2p simply binds to SUMO. The objective of this work is to identify novel signal transduction mechanisms that regulate cytoskeletal networks in response to simulated microgravity conditions. The rationale is that by achieving a fuller understanding of the regulation of cytoskeletal polymers under simulated microgravity conditions on Earth, researchers can develop more complete hypotheses of how the cytoskeleton responds to gravitational changes in the space environment, which can be tested in future space missions. This knowledge may be used to design interventions for adverse health effects that are associated with space travel. The specific goal of this proposal is to determine how simulated microgravity alters the interaction of SUMO with the cellular proteome, and microtubule-associated proteins (MAPS) in particular. The central hypothesis of this work is that microgravity alters the post-translational modifications of lysine residues of a wide variety of cytoskeletal proteins. To test this hypothesis, the post-translational modifications on lysines residues will be analyzed on a proteome-wide basis by mass-spectrometry and immunoblotting under simulated microgravity conditions. We will also test whether the proteome binds differentially to SUMO when it experiences microgravity. An additional emphasis will be placed on the MAP Stu2p.
The Miller lab recently established that the microtubule-associated protein Stu2 interacts with SUMO. Our preliminary data suggest that there are two modes of interaction: a covalent–conjugation with SUMO and a non-covalent interaction. Our long-term goal is to understand how signal transduction circuits control microtubules so that effective and targeted interventions can be developed to manipulate microtubule
functions in stressful situations like disease and space travel. The objective of this work is to identify novel signal transduction mechanisms that regulate the cytoskeleton in response to microgravity conditions. Our central hypothesis is that the previously observed dysfunction of the cytoskeleton under microgravity conditions is mediated by sumoylation. This makes the prediction that SUMO levels will change under simulated microgravity conditions. To test this prediction, we will determine how simulated microgravity alters the interaction of SUMO with the cellular proteome and a microtubule
-associated protein (MAP) in particular. We will complete the following two Aims:
Aim 1. Determine the extent to which sumoylation of proteins in the cell is altered by simulated microgravity. Our working hypothesis is that microgravity alters SUMO conjugation to target lysines in microtubule-associated proteins and the proteome. We will use western blotting and proteomics to assay the extent to which differences in sumoylation are observed in the proteome globally and in Stu2 specifically by simulated
Aim 2. Determine the extent to which simulated microgravity alters the noncovalent binding of cellular proteins to SUMO. Using two forms of SUMO that cannot be conjugated to target proteins, we will treat cells with simulated microgravity conditions and assay the binding of cellular proteins to non-covalent SUMO by proteomic analysis. We will also test the ability of specific cytoskeletal proteins to bind non-covalently to SUMO by western blotting.
This experiment is currently in progress. Results will be available at the conclusion of the study.