Discipline: Chemistry and Chemical Sciences
Subcategory: Biochemistry (not Cell and Molecular Biology and Genetics)
Sara A. Torres Robles - San Diego State University
Co-Author(s): Perla A. Pe'a Palomino, San Diego State University, CA; Sanford I. Bernstein, San Diego State University, CA; Tom Huxford, San Diego State University, CA
Muscle function depends on the assembly of myosin molecules into thick filaments and organization of the sarcomere. Genetic studies have shown in a variety of species that the unc-45 (uncoordinated mutant number 45) gene encodes for a protein, UNC-45, that is essential for the proper assembly, folding, and function of myosin thick filaments in developing skeletal muscle. Subsequent studies identified UNC-45 as being part of a family of UCS (UNC-45, Cro1p, She4p) proteins that are required in diverse myosin-dependent cellular processes. The UNC-45 proteins contain an amino-terminal tetratricopeptide repeat (TPR) domain and a canonical UCS domain. The TPR domain has been identified to interact with the ATP-dependent molecular chaperone Hsp90 (heat shock protein 90). Nevertheless, glutathione S-transferase (GST) pull-down assays have suggested that there is no competition of the TPR domain with UNC-45 for Hsp83, the Drosphila melanogaster homolog of Hsp90. Furthermore, the consequences of the interaction between UNC-45 and Hsp90 proteins on the assembly and function of myosin remain unclear. In order to improve understanding of the UNC-45:Hsp90 protein-protein interaction, we used hydrogen-deuterium exchange-mass spectrometry (HDX-MS). Purified, recombinant UNC-45 and Hsp83 proteins from the fruit fly, Drosphila melanogaster, were prepared and subjected to HDX-MS. The analyses include UNC-45 alone, Hsp83 alone, and UNC-45 with Hsp83. Each analysis was conducted in triplicate at five different D2O exposure times. With the data collected from these analyses we were able to obtain the relative uptake difference for both UNC-45 and Hsp83, which can provide insight into the protein-protein interaction and protein conformational changes upon interaction. We constructed protein models using computational software to model the interaction. The results revealed that when UNC-45 is in the presence of Hsp83, the TPR and, to an even greater extent, the central domain experienced a decrease in deuterium uptake. This suggests that UNC-45 relies upon more than the TPR domain to interact with Hsp83. In addition, the data suggest that Hsp83 experiences significant protein conformational changes in the presence of UNC-45. Further research involves understanding the UNC-45 and Hsp90 protein interactions to obtain a greater understanding of its role in myosin thick filament assembly, as well as molecular mechanisms involved in muscle development and repair.
Funder Acknowledgement(s): Perla Pe'a Palomino; Dr. Jamie Schiffer; Dr. Tom Huxford and the Huxford laboratory; Dr. Sanford Bernstein and the Bernstein laboratory; SDSU MARC program and staff; CSU-LSAMP Senior Alliance NSF Grant HRD-1302873; NIH through the Initiatives for Maximizing Student Opportunities Program ? Grant # 5 R25 GMO58906-16
Faculty Advisor: Dr. Tom Huxford, firstname.lastname@example.org
Role: I am involved in analyzing and interpreting the hydrogen-deuterium exchange-mass spectrometry (HDX-MS) data by using software packages such as DynamX 3.0 Waters software, Excel, R program, and PyMOL. I constructed figures and protein models using the computational software to model possible protein-protein interaction sites and conformational changes obtained from the HDX-MS data.