Download Humans are living significantly longer than ever before, and

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unpublished data, 2016).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
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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.
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http://doi.org/10.1016/j.cell.2013.11.047
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Liu, Y., Po-Min C., & Kuen-Jer, T. (2013). Disease Animal Models of TDP-43 Proteinopathy and Their Pre-Clinical Applications. Int.
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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