Julianne H Grose, Microbiology and Molecular Biology
Evaluation of how well the academic objectives of the proposal were met
- Specific Aim 1: Perform yeast two-hybrid screens on wild type CryAB, R120G CryAB, and HspB2 in order to create a protein-protein interaction network and identify potential substrates.
- Achievements: Large-scale yeast-two hybrid screens were performed on wild type CryAB, R120G CryAB and HspB2 in order to identify their protein-protein interaction networks and to identify putative substrates.
- Specific Aim 2: Validate the protein-protein interaction network via Y2H dependency tests (A), affinity purification from mammalian cells (B) and chaperone activity assays (C).
- Achievements: Y2H dependency tests were performed for all three proteins (CryAB, R120G and HspB2), and a subset of the interactomes was verified through both in vitro (B) and in vivo chaperone assays (C).
Evaluation of the mentoring environment
The eleven students involved learned basic experimental design (with positive and negative controls), troubleshooting, a wide variety of laboratory techniques, and data collection, analysis and presentations skills. In addition, students learned to read and analyze scientific work from other labs since they are given scientific articles to read beginning on their first day in the lab. This project is in collaboration with Ivor Benjamin M.D., Ph.D. at the University of Utah, which allowed these students the unique opportunity of discussing their research with a larger audience once a month.
In addition to intellectual enrichment, students learned to function as a team, with those more experienced (graduate students Kelsey Langston and Whitney Hayes) mentoring those who are new in the lab. As students progressed in their understanding of the project and mastery of basic laboratory skills, they become involved in planning our weekly group meeting, preparing figures for presentations/publications, and writing abstracts/grants/research proposals. For example, graduate students Kelsey Langston and Whitney Hayes prepared figures and wrote-up methods/results for the three scientific articles (one provisionally accepted and the other two in preparation), along with advanced undergraduates Kendall Kiser, Kent Jarvis and Steve Van de Graff. In addition, they were put in charge of particular aspects of the project, such as organizing an Y2H screen in which other undergraduates aided in data collection. I was personally involved in the training of all lab students and met with them once a week to discuss goals, troubleshoot obstacles and analyze data. I prepared a series of training protocols for keeping a notebook, using pipettemen, and all common laboratory techniques. In addition, I aided students in learning these techniques in the lab.
All of these students are progressing in their academic goals, as noted in the following section.
List of students who participated and what academic deliverables they have produced or it is anticipated they will produce
Graduate students (2):
- Whitney Hayes- second year PhD student
- Kelsey Langston – graduated with her Master’s degree in December of 2013
Undergraduate students (9):
- Makay Chapman (continuing as a research assistant)
- Casey Cutler (in medical school)
- Kent Jarvis (in medical school)
- Kendall Kiser (continuing as a research assistant)
- Fred Nelson (in medical school)
- Jonathan Wood (in medical school)
- Katherine Price (in graduate school)
- Jonathan Rice (in medical school)
- Steve Van deGraff (in medical school)
Presentations (students are in bold)
- Hayes, WH, Langston, K, and Grose, JH. (2014) Characterization of Disease-associated HspB2 and CryAB Variants Reveals Chaperone Dysfunction. Poster presentation. ASM Intermountain Branch Meeting, BYU, Provo, UT.
- Hayes, W, Langston, KT, Neubert, J, Benjamin, IJ, and Grose, JH. (2013) Characterizing the Role of HSPB2 and CRYAB in Cardiac Metabolism and Muscle Structure. Poster presentation. Analytical Genetics Meeting Alta, UT.
- Price, K, Chapman, K, Cutler, C, Hayes, W, Lee, S, Louis, K , Nguyen J, and Grose, JH. (2012) Molecular Mechanisms of R120G CryAB-induced Cardiomyopathy. Poster presentation. National Conference for Undergraduate Research (NCUR), Weber State University, UT.
- Rice, J, Neubert, J, Langson, K, Nelson, F, Wood, J, and Grose, JH. (2012) Characterizing the Role of HspB2 in Cardiac Mitochondrial Function. Poster presentation. National Conference for Undergraduate Research (NCUR), Weber State University, UT
Publications (provisionally accepted or in preparation)
- Julianne H. Grose, Xiaohui Wang, Shane Squires, Kelsey Langston, Soumyajit Banerjee Mustafi, Jonathan Neubert, Whitney Hayes, Susan K. Fischer, Matthew Fasano, Gina Moore Saunders, E. Douglas Lewandowski, Elisabeth Christians, Peipei Ping, and Ivor J. Benjamin. Characterization of the Cardiac Overexpression of HspB2 Reveals Mitochondrial and Muscle Roles Supported by a Cardiac HspB2 Interactome. Provisional Acceptance
- Julianne H. Grose, Kelsey Langston and Ivor J. Benjamin. Turning up the heat: heat stress, heat shock proteins, and mitochondrial function. Invited review. In preparation (planned submission December 2014).
- Whitney Hayes, Kendal Kiser, Kent Jarvis, Steve Van deGraff, Keoni Kauwe, Ivor J. Benjamin, and Julianne H. Grose. A CryAB Interactome Reveals Clientele Specificity and Dysfunction Of Mutants Associated With Human Disease In preparation (planned submission January 2015).
Description of the results/findings of the project
The results from the protein binding partner screens demonstrated a separate but overlapping clientele for all three heat shock proteins, CryAB, R120GCryAB and HspB2. This comprises the broadest, and most comprehensive data to show that these chaperones have different clientele specificity. Historically, these proteins were thought to bind virtually every misfolded protein and to have differential functions due to differential tissue expression, cellular localization, etc. Our results suggest that HspB2 has a mitochondrial function, having both mitochondrial and myofibril clientele, while CryAB has mostly myofibril clientele. Their clientele overlap by ~30%, supporting their partially redundant roles as determined by mouse knockout studies. In addition, we confirmed a subset of the binding partners as substrates in both in vitro and in vivo chaperone assays. We were also able to characterize several disease-associated alleles of CryAB, including the well-studied R120G allele. The R120G mutant protein had increased affinity for CryAB binding partners, a possible explanation for its “gain of function” phenotype in humans and mice. Our results not only increase our understanding of the molecular function of this important family of heat shock proteins, but provide assays for characterizing the dysfunction of disease alleles.
Description of how the budget was spent
- ~$10,000 5 undergraduate students for 15 weeks, 8 hours per week at $8.25-10 per hour. This pay was for months in which these students will not be enrolled in the research for credit course 494R. I chose undergraduates to be paid based on seniority in the lab and the number of hours per week they had been volunteering.
- ~$9,800 Research expenses include laboratory consumables, reagents and supplies for wet lab research.
- $800- DNA extraction and amplification and modification
- $2000- yeast and bacterial media, antibiotics and supplies
- $2000- protein purification supplies
- $1000- DNA sequencing and protein mass spectrometry
- $1000- gloves, pipettes, kim-wipes, parafilm, and other consumables
- ~$200 Poster fees and transportation for the 9 undergraduates and 2 graduate students to local scientific conferences.
***The above funding was supplemented with an NIH subaward to Julianne Grose from an NIH Pioneer Grant awarded to Ivor J. Benjamin as well as student travel funds from the Department of Microbiology and Molecular Biology.