M L Spatara

Publications (3)5.01 Total impact

• Article: Transgenic mouse and cell culture models demonstrate a lack of mechanistic connection between endoplasmic reticulum stress and tau dysfunction.

  M L Spatara, A S Robinson
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  ABSTRACT: In vivo aggregation of tau protein is a hallmark of many neurodegenerative disorders, including Alzheimer's disease (AD). Recent evidence has also demonstrated activation of the unfolded protein response (UPR), a cellular response to endoplasmic reticulum (ER) stress, in AD, although the role of the UPR in disease pathogenesis is not known. Here, three model systems were used to determine whether a direct mechanistic link could be demonstrated between tau aggregation and the UPR. The first model system used was SH-SY5Y cells, a neuronal cultured cell line that endogenously expresses tau. In this system, the UPR was activated using chemical stressors, tunicamycin and thapsigargin, but no changes in tau expression levels, solubility, or phosphorylation were observed. In the second model system, wild-type 4R tau and P301L tau, a variant with increased aggregation propensity, were heterologously overexpressed in HEK 293 cells. This overexpression did not activate the UPR. The last model system examined here was the PS19 transgenic mouse model. Although PS19 mice, which express the P301S variant of tau, display severe neurodegeneration and formation of tau aggregates, brain tissue samples did not show any activation of the UPR. Taken together, the results from these three model systems suggest that a direct mechanistic link does not exist between tau aggregation and the UPR.
  Journal of Neuroscience Research 02/2010; 88(9):1951-61. · 2.74 Impact Factor
• Article: Kinetic folding studies of the P22 tailspike beta-helix domain reveal multiple unfolded states.

  M L Spatara, C J Roberts, A S Robinson
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  ABSTRACT: The beta-helix is an important protein fold in many pathogens, and is a challenging system for folding pathway prediction because it primarily is stabilized by non-local interactions along the primary sequence. A useful experimental model of this fold is a monomeric truncation of P22 tailspike protein, the beta-helix domain (bhx). This report describes a systematic in vitro study of the chemical denaturation and refolding of bhx. Results from equilibrium chemical denaturation experiments were consistent with a two-state folding mechanism, but showed only partial reversibility. Stopped-flow fluorescence studies showed a single unfolding step, but two refolding steps. The slow refolding step could be partly attributed to proline isomerization, based on an increased rate during refolding in the presence of PPIase and an increased relative amplitude of this step with increasing delay time in double-jump refolding experiments observed over delays of 5-100 s. However, double-jump refolding experiments with delay times longer than 100 s along with size exclusion chromatography and dynamic light scattering of refolding samples showed that the overall refolding yield decreased as bhx was unfolded for longer periods of time. Furthermore, the losses resulted from aggregate formation during refolding. This suggests that a change occurs over time in the unfolded or denatured state ensemble that increases the propensity for aggregation upon the shift to more native-favoring conditions. Alternatively aggregate nuclei may be able to form even under high denaturant conditions, and these subsequently grow and consume monomer when placed under native-favoring conditions.
  Biophysical chemistry 06/2009; 141(2-3):214-21. · 2.28 Impact Factor
• Article: Kinetic folding studies of the P22 tailspike beta-helix domain reveal multiple unfolded states

  M.L. Spatara, C.J. Roberts, A.S. Robinson
  [show abstract] [hide abstract]
  ABSTRACT: The beta-helix is an important protein fold in many pathogens, and is a challenging system for folding pathway prediction because it primarily is stabilized by non-local interactions along the primary sequence. A useful experimental model of this fold is a monomeric truncation of P22 tailspike protein, the beta-helix domain (bhx). This report describes a systematic invitro study of the chemical denaturation and refolding of bhx. Results from equilibrium chemical denaturation experiments were consistent with a two-state folding mechanism, but showed only partial reversibility. Stopped-flow fluorescence studies showed a single unfolding step, but two refolding steps. The slow refolding step could be partly attributed to proline isomerization, based on an increased rate during refolding in the presence of PPIase and an increased relative amplitude of this step with increasing delay time in double-jump refolding experiments observed over delays of 5–100 s. However, double-jump refolding experiments with delay times longer than 100 s along with size exclusion chromatography and dynamic light scattering of refolding samples showed that the overall refolding yield decreased as bhx was unfolded for longer periods of time. Furthermore, the losses resulted from aggregate formation during refolding. This suggests that a change occurs over time in the unfolded or denatured state ensemble that increases the propensity for aggregation upon the shift to more native-favoring conditions. Alternatively aggregate nuclei may be able to form even under high denaturant conditions, and these subsequently grow and consume monomer when placed under native-favoring conditions.
  Biophysical Chemistry.