The Effect of Parkinson's-Disease-Associated Mutations on the Deubiquitinating Enzyme UCH-L1

Andersson, Fredrik I.; Werrell, Elizabeth F.; McMorran, Lindsay M.; Crone, William J. K.; Das, Chittarnajan; Hsu, Shang-Te Danny; Jackson, Sophie

doi:10.1016/j.jmb.2010.12.029

Cited by 64 publications

(104 citation statements)

References 35 publications

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“…The rate constants associated with the evolution of each unfolding phase measured in the interrupted-refolding experiments are shown as open or filled diamonds with their associated fitting errors displayed. Wild-type data were taken from an earlier investigation (28). To see this figure in color, go online.…”

Section: Results From Single-[urea]-jump Unfolding and Refolding Expementioning

confidence: 99%

“…WT and variant UCH-L1 were purified as described elsewhere (28). The concentration of the purified protein was determined by spectrophotometry using an extinction coefficient of 28,086 M À1 cm À1 at 280 nm.…”

Section: Protein Expression and Purificationmentioning

confidence: 99%

“…The conformational stabilities of the WT and variants were measured using urea unfolding experiments conducted under equilibrium conditions in which it has been established that the unfolding of WT UCH-L1 is fully reversible (28). Unfolding was monitored by Trp fluorescence and far-UV CD spectroscopy to probe changes in the tertiary and secondary models, respectively.…”

Section: Kinetic and Thermodynamic Measurements On Folding And Unfoldingmentioning

confidence: 99%

“…Researchers have investigated wildtype (WT) UCH-L1 using a range of different techniques. For example, the fluorescence of the single tryptophan, Trp26, was used to probe changes in tertiary structure in both thermodynamic and kinetic studies (28,29). These studies showed that UCH-L1 unfolds reversibly under equilibrium conditions using urea as a denaturant, and that at least one stable intermediate state (I) is populated between the native (N) and denatured (D) states (28).…”

Section: Introductionmentioning

confidence: 99%

“…For example, the fluorescence of the single tryptophan, Trp26, was used to probe changes in tertiary structure in both thermodynamic and kinetic studies (28,29). These studies showed that UCH-L1 unfolds reversibly under equilibrium conditions using urea as a denaturant, and that at least one stable intermediate state (I) is populated between the native (N) and denatured (D) states (28). More recently, kinetic studies established that WT UCH-L1 folds along parallel pathways, each of which possesses a detectable intermediate state (I 1 or I 2 ) (29), as observed for UCH-L3.…”

Section: Introductionmentioning

confidence: 99%

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Characterization of the Folding of a 5 2 -Knotted Protein Using Engineered Single-Tryptophan Variants

Zhang

Jackson

2016

Biophysical Journal

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An increasing number of proteins that contain topological knots have been identified over the past two decades; however, their folding mechanisms are still not well understood. UCH-L1 has a 5 2 -knotted topology. Here, we constructed a series of variants that contain a single tryptophan at different locations along the polypeptide chain. A study of the thermodynamic properties of the variants shows that the structure of UCH-L1 is remarkably tolerant to incorporation of bulky tryptophan side chains. Comprehensive kinetic studies of the variants reveal that they fold via parallel pathways on which there are two intermediate states very similar to wild-type UCH-L1. The structures of the intermediate states do not change significantly with mutation and therefore occupy local minima on the energy landscape that have relatively narrow basins. The kinetic studies also establish that there are considerably more tertiary interactions in the intermediate states than results from previous NMR studies suggested. The two intermediates differ from each other in the extent to which tertiary interactions between the highly stable central b-sheet and flanking a-helices and loop regions are formed. There is strong evidence that these states are aggregation prone. The transition states from both I 1 and I 2 to the native state are plastic and change with mutation and denaturant concentration. Previous studies indicated that the threading event that creates the 5 2 knot occurs during these steps, suggesting that there is a broad energy barrier consistent with the chain undergoing some searching of conformational space as the threading takes place.

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Section: Results From Single-[urea]-jump Unfolding and Refolding Expementioning

confidence: 99%

Section: Protein Expression and Purificationmentioning

confidence: 99%

Section: Kinetic and Thermodynamic Measurements On Folding And Unfoldingmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Characterization of the Folding of a 5 2 -Knotted Protein Using Engineered Single-Tryptophan Variants

Zhang

Jackson

2016

Biophysical Journal

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Ubiquitin C‐Terminal Hydrolase L1: Biochemical and Cellular Characterization of a Covalent Cyanopyrrolidine‐Based Inhibitor

Krabill¹,

Chen²,

Hussain

et al. 2019

ChemBioChem

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The deubiquitinase (DUB) ubiquitin C‐terminal hydrolase L1 (UCHL1) is expressed primarily in the central nervous system under normal physiological conditions. However, UCHL1 is overexpressed in various aggressive forms of cancer with strong evidence supporting UCHL1 as an oncogene in lung, glioma, and blood cancers. In particular, the level of UCHL1 expression in these cancers correlates with increased invasiveness and metastatic behavior, as well as poor patient prognosis. Although UCHL1 is considered an oncogene with potential as a therapeutic target, there remains a significant lack of useful small‐molecule probes to pharmacologically validate in vivo targeting of the enzyme. Herein, we describe the characterization of a new covalent cyanopyrrolidine‐based UCHL1 inhibitory scaffold in biochemical and cellular studies to better understand the utility of this inhibitor in elucidating the role of UCHL1 in cancer biology.

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Transient sampling of aggregation‐prone conformations causes pathogenic instability of a parkinsonian mutant of DJ‐1 at physiological temperature

et al. 2015

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Various missense mutations in the cytoprotective protein DJ-1 cause rare forms of inherited parkinsonism. One mutation, M26I, diminishes DJ-1 protein levels in the cell but does not result in large changes in the three-dimensional structure or thermal stability of the protein. Therefore, the molecular defect that results in loss of M26I DJ-1 protective function is unclear. Using NMR spectroscopy near physiological temperature, we found that the picosecond-nanosecond dynamics of wild-type and M26I DJ-1 are similar. In contrast, elevated amide hydrogen/deuterium exchange rates indicate that M26I DJ-1 is more flexible than the wild-type protein on longer timescales and that hydrophobic regions of M26I DJ-1 are transiently exposed to solvent. Tryptophan fluorescence spectroscopy and thiol crosslinking analyzed by mass spectrometry also demonstrate that M26I DJ-1 samples conformations that differ from the wild-type protein at 378C. These transiently sampled conformations are unstable and cause M26I DJ-1 to aggregate in vitro at physiological temperature but not at lower temperatures. M26I DJ-1 aggregation is correlated with pathogenicity, as the structurally similar but non-pathogenic M26L mutation does not aggregate at 378C. The onset of dynamically driven M26I DJ-1 instability at physiological temperature resolves conflicting literature reports about the behavior of this disease-associated mutant and illustrates the pitfalls of characterizing proteins exclusively at room temperature or below, as key aspects of their behavior may not be apparent.

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The Effect of Parkinson's-Disease-Associated Mutations on the Deubiquitinating Enzyme UCH-L1

Cited by 64 publications

References 35 publications

Characterization of the Folding of a 5 2 -Knotted Protein Using Engineered Single-Tryptophan Variants

Characterization of the Folding of a 5 2 -Knotted Protein Using Engineered Single-Tryptophan Variants

Ubiquitin C‐Terminal Hydrolase L1: Biochemical and Cellular Characterization of a Covalent Cyanopyrrolidine‐Based Inhibitor

Transient sampling of aggregation‐prone conformations causes pathogenic instability of a parkinsonian mutant of DJ‐1 at physiological temperature

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