On the Design of Broad Based Screening Assays to Identify Potential Pharmacological Chaperones of Protein Misfolding Diseases

Naik, Subhashchandra; Zhang, Na; Gao, Phillip; Fisher, Mark T.

doi:10.2174/1568026611212220006

Cited by 15 publications

(17 citation statements)

References 111 publications

(118 reference statements)

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“…For SOD1, oligomerization and aggregation are proportional to the fraction of monomers (Khare et al, 2004). Researchers have developed various strategies to stabilize SOD1 dimer, including co-expression of chaperones and small molecule binding (Bruening et al, 1999; Limpert et al, 2013; Naik et al, 2012). T2D-SOD1 features a significantly reduce the population of monomers and oligomers, which underlies its beneficial effect to motor neuron-like cells.…”

Section: Discussionmentioning

confidence: 99%

A Phosphomimetic Mutation Stabilizes SOD1 and Rescues Cell Viability in the Context of an ALS-Associated Mutation

et al. 2016

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SUMMARY The majority of amyotrophic lateral sclerosis (ALS)-related mutations in the enzyme Cu, Zn superoxide dismutase (SOD1), as well as a post-translational modification, glutathionylation, destabilize the protein and lead to a misfolded oligomer that is toxic to motor neurons. The biophysical role of another physiological SOD1 modification, T2-phosphorylation, has remained a mystery. Here, we find that a phosphomimetic mutation, T2D, thermodynamically stabilizes SOD1 even in the context of a strongly SOD1-destabilizing mutation, A4V, one of the most prevalent and aggressive ALS-associated mutations in North America. This stabilization protects against formation of toxic SOD oligomers and positively impacts motor neuron survival in cellular assays. We solve the crystal structure of T2D-SOD1 and explain its stabilization effect using DMD simulations. These findings imply that T2-phosphorylation may be a plausible innate cellular protection response against SOD1-induced cytotoxicity, and stabilizing the SOD1 native conformation might offer us viable pharmaceutical strategies against currently incurable ALS.

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Section: Discussionmentioning

confidence: 99%

A Phosphomimetic Mutation Stabilizes SOD1 and Rescues Cell Viability in the Context of an ALS-Associated Mutation

et al. 2016

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“…They monitored chaperonin binding to partially folded protein substrates attached on immobilized surfaces to eventually follow the kinetics of the ATP/GroES induced release of the GroEL chaperonin from the surface 35, 36 . If the dynamic folding equilibria of a protein results in substantial populations of metastable folded states, this particular immobilization approach can also be used to examine possible ligand or solution induced stabilization shift away from the metastable population 14 .…”

Section: Resultsmentioning

confidence: 99%

“…14 For example, generation of smaller aggregates reduce the light scattering signal yielding false positive results. Furthermore, protein aggregation based assays are notorious for their slow kinetic readouts, particularly during oligomer dissociation processes.…”

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confidence: 99%

Chaperonin-Based Biolayer Interferometry To Assess the Kinetic Stability of Metastable, Aggregation-Prone Proteins

et al. 2016

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Stabilizing the folded state of metastable and/or aggregation-prone proteins through exogenous ligand binding is an appealing strategy to decrease disease pathologies brought on by protein folding defects or deleterious kinetic transitions. Current methods of examining ligand binding to these marginally stable native states are limited, because protein aggregation typically interferes with analysis. Here, we describe a rapid method for assessing the kinetic stability of folded proteins and monitoring the effects of ligand stabilization for both intrinsically stable proteins (monomers, oligomers, multi-domain) and metastable proteins (e.g. low Tm) that uses a new GroEL chaperonin-based biolayer interferometry (BLI) denaturant-pulse platform. A kinetically controlled denaturation isotherm is generated by exposing a target protein immobilized on a BLI biosensor to increasing denaturant concentrations (urea or GnHCl) in a pulsatile manner to induce partial or complete unfolding of the attached protein population. Following the rapid removal of the denaturant, the extent of hydrophobic unfolded/partially folded species that remain is detected by increased GroEL binding. Since this kinetic denaturant pulse is brief, the amplitude of the GroEL binding to the immobilized protein depends on the duration of exposure to denaturant, the concentration of denaturant, wash times, and the underlying protein unfolding/refolding kinetics; fixing all other parameters and plotting GroEL binding amplitude versus denaturant pulse concentration results in a kinetically controlled denaturation isotherm. When folding osmolytes or stabilizing ligands are added to the immobilized target proteins before and during the denaturant pulse, the diminished population of unfolded/partially folded protein is manifested by a decreased GroEL binding and/or a marked shift in these kinetically controlled denaturation profiles to higher denaturant concentrations. This particular platform approach can be used to identify small molecules/solution conditions that can stabilize or destabilize thermally stable proteins, multi-domain proteins, oligomeric proteins, and most importantly, aggregation prone metastable proteins.

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“…, the nucleotide‐free GroEL has the tightest binding affinity for partially folded or hydrophobic protein, is stable, binding is reversible, the binding site is large, and the binding is promiscuous. Any conformational alteration and destabilization in the structures of certain proteins in solution can also be identified by this same method . This approach also works with a reverse‐oriented (GroEL on the biosensor) BLI method .…”

Section: Discussionmentioning

confidence: 99%

“…). Identification of structurally altered and aggregated states of proteins using GroEL as a probe on bio‐layer interferometry has been previously described and validated with SPR . Chaperone binding to immobilized protein and ATP‐mediated chaperone release can be monitored on bio‐layer interferometry similar to previously done work on surface plasmon resonance .…”

Section: Methodsmentioning

confidence: 99%

Protein folding on biosensor tips: folding of maltodextrin glucosidase monitored by its interactions with GroEL

et al. 2016

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Protein folding has been extensively studied for past four decades by employing solution based experiments such as solubility, enzymatic activity, secondary structure analysis, and analytical methods like FRET, NMR and HD exchange. However, for rapid analysis of the folding process, solution based approaches are often plagued with aggregation side reactions resulting in poor yields. In this work we demonstrate that a Bio-Layer Interferometry (BLI) chaperonin detection system can be potentially applied to identify superior refolding conditions for denatured proteins. The degree of immobilized protein folding as a function of time can be detected by monitoring the binding of the high-affinity nucleotide-free form of the chaperonin GroEL. GroEL preferentially interacts with proteins that have hydrophobic surfaces exposed in their unfolded or partially folded form so a decrease in GroEL binding can be correlated with burial of hydrophobic surfaces as folding progresses. The magnitude of GroEL binding to the protein immobilized on Bio-layer interferometry biosensor inversely reflects the extent of protein folding and hydrophobic residue burial. We demonstrate conditions where accelerated folding can be observed for the aggregation prone protein Maltodextrin glucosidase (MalZ). Superior immobilized folding conditions identified on the Bio-layer interferometry biosensor surface were reproduced on Ni-NTA sepharose bead surfaces and resulted in significant improvement in folding yields of released MalZ (measured by enzymatic activity) compared to bulk refolding conditions in solution.

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On the Design of Broad Based Screening Assays to Identify Potential Pharmacological Chaperones of Protein Misfolding Diseases

Cited by 15 publications

References 111 publications

A Phosphomimetic Mutation Stabilizes SOD1 and Rescues Cell Viability in the Context of an ALS-Associated Mutation

A Phosphomimetic Mutation Stabilizes SOD1 and Rescues Cell Viability in the Context of an ALS-Associated Mutation

Chaperonin-Based Biolayer Interferometry To Assess the Kinetic Stability of Metastable, Aggregation-Prone Proteins

Protein folding on biosensor tips: folding of maltodextrin glucosidase monitored by its interactions with GroEL

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