The central domain of <scp>UNC</scp>‐45 chaperone inhibits the myosin power stroke

Bujalowski, Paul J.; Nicholls, Paul; Garza, Eleno; Oberhauser, Andrés F.

doi:10.1002/2211-5463.12346

Cited by 17 publications

(12 citation statements)

References 29 publications

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“…These processes underlie many important phenomena such as those related to cellular homeostasis [ 9 , 10 , 11 , 12 , 13 ], cancer metabolism [ 1 , 14 , 15 , 16 , 17 ], and the onset and treatment of protein misfolding diseases [ 1 , 18 , 19 , 20 ]. Single-molecule force spectroscopy (SMFS) techniques are particularly powerful tools for studying protein folding because they provide a means to directly apply forces to individual proteins under native conditions in order to measure their structural response and the internal forces that stabilize the protein [ 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , ...…”

Section: Introductionmentioning

confidence: 99%

Mechanical Stability of a Small, Highly-Luminescent Engineered Protein NanoLuc

Ding

Apostolidou

Marszałek

2020

IJMS

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NanoLuc is a bioluminescent protein recently engineered for applications in molecular imaging and cellular reporter assays. Compared to other bioluminescent proteins used for these applications, like Firefly Luciferase and Renilla Luciferase, it is ~150 times brighter, more thermally stable, and smaller. Yet, no information is known with regards to its mechanical properties, which could introduce a new set of applications for this unique protein, such as a novel biomaterial or as a substrate for protein activity/refolding assays. Here, we generated a synthetic NanoLuc derivative protein that consists of three connected NanoLuc proteins flanked by two human titin I91 domains on each side and present our mechanical studies at the single molecule level by performing Single Molecule Force Spectroscopy (SMFS) measurements. Our results show each NanoLuc repeat in the derivative behaves as a single domain protein, with a single unfolding event occurring on average when approximately 72 pN is applied to the protein. Additionally, we performed cyclic measurements, where the forces applied to a single protein were cyclically raised then lowered to allow the protein the opportunity to refold: we observed the protein was able to refold to its correct structure after mechanical denaturation only 16.9% of the time, while another 26.9% of the time there was evidence of protein misfolding to a potentially non-functional conformation. These results show that NanoLuc is a mechanically moderately weak protein that is unable to robustly refold itself correctly when stretch-denatured, which makes it an attractive model for future protein folding and misfolding studies.

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Section: Introductionmentioning

confidence: 99%

Mechanical Stability of a Small, Highly-Luminescent Engineered Protein NanoLuc

Ding

Apostolidou

Marszałek

2020

IJMS

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“…The basic mechanism in muscle involves the interaction of the protein filaments myosin and actin, both of which are affected by mutations in Hsp90 cochaperones. The TPR-containing cochaperones encoded by UNC45A and UNC45B promote myosin folding ( Lee et al, 2014 ; Lehtimaki et al, 2017 ; Bujalowski et al, 2018 ). Mutations that alter the myosin-binding domain of UNC45B were found in a patient with congenital muscle disorder ( Dafsari et al, 2019 ).…”

Section: Heart and Muscle Disordersmentioning

confidence: 99%

Mutations in Hsp90 Cochaperones Result in a Wide Variety of Human Disorders

Johnson

2021

Front. Mol. Biosci.

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The Hsp90 molecular chaperone, along with a set of approximately 50 cochaperones, mediates the folding and activation of hundreds of cellular proteins in an ATP-dependent cycle. Cochaperones differ in how they interact with Hsp90 and their ability to modulate ATPase activity of Hsp90. Cochaperones often compete for the same binding site on Hsp90, and changes in levels of cochaperone expression that occur during neurodegeneration, cancer, or aging may result in altered Hsp90-cochaperone complexes and client activity. This review summarizes information about loss-of-function mutations of individual cochaperones and discusses the overall association of cochaperone alterations with a broad range of diseases. Cochaperone mutations result in ciliary or muscle defects, neurological development or degeneration disorders, and other disorders. In many cases, diseases were linked to defects in established cochaperone-client interactions. A better understanding of the functional consequences of defective cochaperones will provide new insights into their functions and may lead to specialized approaches to modulate Hsp90 functions and treat some of these human disorders.

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“…Based upon actin filament gliding assays of myosin heads with and without the presence of UNC-45 and/or HSP-90 (13,14), the following model can be envisioned:…”

Section: Introductionmentioning

confidence: 99%

Mutations in conserved residues of the myosin chaperone UNC-45 result in both reduced stability and chaperoning activity

Moncrief

Matheny

Gaziova

et al. 2021

Preprint

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Proper muscle development and function depends on myosin being properly folded and integrated into the thick filament structure. For this to occur the myosin chaperone UNC-45, or UNC-45B, must be present and able to chaperone myosin. Here we use a combination of in vivo C. elegans experiments and in vitro biophysical experiments to analyze the effects of six missense mutations in conserved regions of UNC-45/UNC-45B. We found that the phenotype of paralysis and disorganized thick filaments in 5/6 of the mutant nematode strains can likely be attributed to both reduced steady state UNC-45 protein levels and reduced chaperone activity. Interestingly, the biophysical assays performed on purified proteins show that all of the mutations result in reduced myosin chaperone activity but not overall protein stability. This suggests that these mutations only cause protein instability in the in vivo setting and that these conserved regions may be involved in UNC-45 protein stability/ regulation via post translational modifications, protein-protein interactions, or some other unknown mechanism.

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The central domain of UNC‐45 chaperone inhibits the myosin power stroke

Cited by 17 publications

References 29 publications

Mechanical Stability of a Small, Highly-Luminescent Engineered Protein NanoLuc

Mechanical Stability of a Small, Highly-Luminescent Engineered Protein NanoLuc

Mutations in Hsp90 Cochaperones Result in a Wide Variety of Human Disorders

Mutations in conserved residues of the myosin chaperone UNC-45 result in both reduced stability and chaperoning activity

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