* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Humans are living significantly longer than ever before, and
Survey
Document related concepts
Homology modeling wikipedia , lookup
Bimolecular fluorescence complementation wikipedia , lookup
Protein structure prediction wikipedia , lookup
Protein folding wikipedia , lookup
Protein domain wikipedia , lookup
Protein mass spectrometry wikipedia , lookup
Western blot wikipedia , lookup
Protein purification wikipedia , lookup
Nuclear magnetic resonance spectroscopy of proteins wikipedia , lookup
List of types of proteins wikipedia , lookup
Transcript
Name: Oscar Hernandez Murillo PI’s Name: James Shorter University Scholars Research Progress Report Engineering α-synuclein-specific Hsp104 variants using signatures of functional divergence Humans are living significantly longer than ever before, and diseases related to senescence have become increasingly common placing mammoth social and economic burdens upon our society (Hindle, 2010). These diseases are frequently neurodegenerative disorders, which despite having different clinical manifestations, share a similar pathology: the misfolding and aggregation of disease-specific proteins (Cushman et al., 2010; Liu et al., 2013). Therefore, a great emphasis has been made on possible therapies that can dissolve these aggregates and reduce proteotoxicity in cells (Cushman et al., 2010). Hsp104, a hexameric protein member of the Hsp100/ClpB family of ATPases, is capable of disaggregating amyloid fibers, by itself, and disordered aggregates, with the help of the Hsp70/Hsp40 chaperone system, and returns the aggregated proteins to their native, soluble state in S. cerevisiae (DeSantis, Shorter J., 2012). Since no apparent metazoan homolog has been found, engineering and applying Hsp104 to reverse protein aggregation found in neurodegenerative diseases, such as Parkinson’s disease (PD) and amyotrophic lateral sclerosis (ALS) could lead to possible therapeutic applications (Shorter, 2008; Jackrel, M. & Shorter, J., 2015). Previously isolated potentiated variants of Hsp104 rescue proteotoxicity induced by α-synuclein (found in PD), FUS and TDP-43 (both found in ALS) aggregation in their respective yeast models (Jackrel et al, 2014a; Stefanis, 2012; Philips, T., et al, 2013). Unfortunately, these variants can incur growth defects at 37°C, probably due to off-target effects (Jackrel et al, 2015). Therefore, engineering more substrate-specific variants represents a priority if Hsp104 is to be used to mitigate the effects of protein aggregation in neurodegeneration. As it has recently been found that certain Hsp104 homologs can rescue α-synuclein selectively in yeast, an approach exploiting the functional divergence between Hsp104 and these selective homologs is appealing for the creation of substrate-specific variants (March, Z. unpublished data, 2016). This approach should also take into account possible epistatic interactions (as some phenotypes need permissive mutations to arise) while prioritizing residues making up the inner channel of Hsp104 (as these engage in substrate binding and translocation) (Ortlund et al, 2007; Yokom et al., 2016). Therefore, I hypothesized that substrate-specific Hsp104 variants can be engineered by mutating sets of co-evolving innerchannel residues that are responsible for functional divergence. Using an array of computational tools, I was able to identify potentials sets of co-evolving inner-channel residues responsible for functional divergence between S. cerevisiae Hsp104 and α-synuclein selective homologs. Substitutions at identified residues were then made based on the amino acid present in the sequences of the selective Hsp104 homologs and some of these mutations were tested in yeast models of neurodegenerative diseases. Thus far, one αsynuclein specific variant, Hsp104A249G-A503S, has been confirmed through this process. These results are exciting as they could lead to the discovery of Hsp104 variants with potential applications in neurodegenerative disease therapeutics and purification of aggregation-prone recombinant proteins. References Cushman, M., Johnson, B.S., King, O.D., Gitler, A.D., & Shorter J. (2010). Prion-like disorders: blurring the divide between transmissibility and infectivity. J. Cell Sci., 123, 1191-1201. DeSantis, M.E., & Shorter J. (2012). The elusive middle domain of Hsp104 and ClpB: location and function. Biochimica et Biophysica Acta, 1823(1), 29-39. Hindle, J. V. (2010). Ageing, neurodegeneration and Parkinson’s disease. Age Ageing, 39, 156-161. Jackrel, M. E., & Shorter, J. (2015). Engineering enhanced protein disaggregases for neurodegenerative disease. Prion, 9(2), 90109. Jackrel, M. E., DeSantis, M. E., Martinez, B. A., Castellano, L. M., Stewart, R. M., Caldwell, K. A., Caldwell, G. A. & Shorter, J. (2014). Potentiated Hsp104 variants antagonize diverse proteotoxic misfolding events. Cell, 156(0), 170–182. http://doi.org/10.1016/j.cell.2013.11.047 Jackrel, M. E., Yee, K., Tariq, A., Chen, AI., & Shorter, J (2015). Disparate mutations confer therapeutic gain of Hsp104 function. ACS chemical biology 10(12): 2672-2679. Liu, Y., Po-Min C., & Kuen-Jer, T. (2013). Disease Animal Models of TDP-43 Proteinopathy and Their Pre-Clinical Applications. Int. J. Mol. Sci., 14(10), 20079-20111. Philips, T., Bento-Abreu, A., Nonneman, A., Haeck, W., Staats, K., Geelen, V., Hersmus, N., Küsters, B., Van Den Bosch, L., Van Damme, P., Richardson, W.D., & Robberecht, W. (2013). Oligodendrocyte dysfunction in the pathogenesis of amyotrophic lateral sclerosis. Brain, 136 (2), 473. Shorter, J. (2008). Hsp104: a weapon to combat diverse neurodegenerative disorders. Neurosignal, 16, 63-74. Stefanis, L. (2012) α-Synuclein in Parkinson's Disease. Cold Spring Harb Perspect Med 2 (2): a009399. Ortlund, E. A., Bridgham, J. T., Redinbo, M. R., & Thornton, J. W. (2007). Crystal structure of an ancient protein: evolution by conformational epistasis. Science, 317(5844), 1544-1548. Yokom, A. L., Gates, S. N., Jackrel, M. E., Mack, K. L., Su, M., Shorter, J., & Southworth, D. R. (2016). Spiral architecture of the Hsp104 disaggregase reveals the basis for polypeptide translocation. Nature Structural & Molecular Biology 23, 830–837