Engineering of a Polydisperse Small Heat-Shock Protein Reveals Conserved Motifs of Oligomer Plasticity

Mishra, Sanjay; Chandler, Shane A.; Williams, Dewight; Claxton, Derek; Koteiche, Hanane A.; Stewart, Phoebe L.; Benesch, Justin L. P.; Mchaourab, Hassane S.

doi:10.1101/247288

Cited by 3 publications

(5 citation statements)

References 69 publications

(109 reference statements)

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“…Note that the C137S mutation does not impact the overall structure of the cHSP27 dimer (Alderson et al, 2019). The molecular mass of WT HSP27 is in good agreement with previous studies (Jovcevski et al, 2015; Mishra et al, 2018), with masses of 670 kDa obtained by analytical SEC and 400 kDa by SEC‐MALS. The hydration radii of WT and P182L HSP27, as determined by SEC‐MALS, are 9 and 38 nm, respectively.…”

Section: Methodssupporting

confidence: 91%

A weakened interface in the P182L variant of HSP27 associated with severe Charcot‐Marie‐Tooth neuropathy causes aberrant binding to interacting proteins

et al. 2021

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HSP27 is a human molecular chaperone that forms large, dynamic oligomers and functions in many aspects of cellular homeostasis. Mutations in HSP27 cause Charcot‐Marie‐Tooth (CMT) disease, the most common inherited disorder of the peripheral nervous system. A particularly severe form of CMT disease is triggered by the P182L mutation in the highly conserved IxI/V motif of the disordered C‐terminal region, which interacts weakly with the structured core domain of HSP27. Here, we observed that the P182L mutation disrupts the chaperone activity and significantly increases the size of HSP27 oligomers formed in vivo, including in motor neurons differentiated from CMT patient‐derived stem cells. Using NMR spectroscopy, we determined that the P182L mutation decreases the affinity of the HSP27 IxI/V motif for its own core domain, leaving this binding site more accessible for other IxI/V‐containing proteins. We identified multiple IxI/V‐bearing proteins that bind with higher affinity to the P182L variant due to the increased availability of the IxI/V‐binding site. Our results provide a mechanistic basis for the impact of the P182L mutation on HSP27 and suggest that the IxI/V motif plays an important, regulatory role in modulating protein–protein interactions.

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Section: Methodssupporting

confidence: 91%

A weakened interface in the P182L variant of HSP27 associated with severe Charcot‐Marie‐Tooth neuropathy causes aberrant binding to interacting proteins

et al. 2021

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“…Note that the C137S mutation does not impact the overall structure of the cHSP27 dimer (64). The molecular mass of WT HSP27 is in good agreement with previous publications (84,90), with the former reference indicating 670 kDa by analytical SEC and the latter 400 kDa by SEC-MALS.…”

Section: Sec-mals Smolecular Masses Were Estimated By Analytical Secsupporting

confidence: 90%

“…Alternatively, lowering the affinity of the IxI/V motif for the ACD may allosterically trigger a change in the conformation of the NTD or its inter-subunit interactions. In addition, multiple lines of evidence point to the significance of the NTR in regulating oligomeric assembly (84), some of which contain IxI/V motifs or similar variations thereof (70), with oligomerization still observed for CTR-truncated forms of HSP27, but not NTR-truncated forms (20). In addition, it is interesting to note that mutations in other sHSPs, including α-and γ-crystallin, are linked to diseases such as cardiomyopathy and congenital cataract formation.…”

Section: Discussionmentioning

confidence: 99%

“…Image processing and analysis. Cytoplasmic regions of interest were randomly selected from image stacks (200 x 200 pixels, single slice) and the HSP27 distribution was analyzed using the Fiji distribution of ImageJ [83,84]. After noise filtering (Gaussian blur, sigma 0.7), high intensity spots were detected with the Find Maxima tool (fixed noise level 50).…”

Section: Sec-mals Smolecular Masses Were Estimated By Analytical Secmentioning

confidence: 99%

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Dysregulated interactions triggered by a neuropathy-causing mutation in the IPV motif of HSP27

Alderson

Adriaenssens

Asselbergh

et al. 2019

Preprint

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Molecular chaperone | small heat-shock protein | Charcot-Marie-Tooth disease | short linear motif | nuclear magnetic resonance | protein aggregation | neurological diseaseCorrespondence: vincent.timmerman@uantwerpen.be, andrew.baldwin@chem.ox.ac.uk, justin.benesch@chem.ox.ac.uk the backbone atoms of V111, T113, and L157 (Fig. 1E). In HSP27, the IxI/V motif corresponds to I181-P182-V183; the Ile and Val residues penetrate the β4/β8 groove while P182 does not make any direct contacts with the ACD (Fig. 1E).The P182L variant has impaired chaperone activity in vitro. We purified recombinant human HSP27 and the CMTimplicated P182L variant from E. coli, and compared their chaperone activities in vitro using a substrate aggregation assay. We tested their ability to prevent the aggregation of two model substrate proteins, malate dehydrogenase (MDH) and

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“…First, we identify a direct interaction between tau and a specific region of the NTR, the hydrophobic and proline-rich insertion region that is unique to HspB1 and makes its NTR the longest among human sHSPs (27,28). Our study indirectly implicates a second NTR subregion lying between the insertion region and the start of the structured ACD; mutation of a serine to cysteine in this disordered region greatly reduces tau-binding ability and diminishes chaperone activity.…”

Section: Discussionmentioning

confidence: 86%

Release of a disordered domain enhances HspB1 chaperone activity toward tau

Baughman

Pham

Adams

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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Small heat shock proteins (sHSPs) are a class of ATP-independent molecular chaperones that play vital roles in maintaining protein solubility and preventing aberrant protein aggregation. They form highly dynamic, polydisperse oligomeric ensembles and contain long intrinsically disordered regions. Experimental challenges posed by these properties have greatly impeded our understanding of sHSP structure and mechanism of action. Here we characterize interactions between the human sHSP HspB1 (Hsp27) and microtubule-associated protein tau, which is implicated in multiple dementias, including Alzheimer’s disease. We show that tau binds both to a well-known binding groove within the structured alpha-crystallin domain (ACD) and to sites within the enigmatic, disordered N-terminal region (NTR) of HspB1. However, only interactions involving the NTR lead to productive chaperone activity, whereas ACD binding is uncorrelated with chaperone function. The tau-binding groove in the ACD also binds short hydrophobic regions within HspB1 itself, and HspB1 mutations that disrupt these intrinsic ACD–NTR interactions greatly enhance chaperone activity toward tau. This leads to a mechanism in which the release of the disordered NTR from a binding groove on the ACD enhances chaperone activity toward tau. The study advances understanding of the mechanisms by which sHSPs achieve their chaperone activity against amyloid-forming clients and how cells defend against pathological tau aggregation. Furthermore, the resulting mechanistic model points to ways in which sHSP chaperone activity may be increased, either by native factors within the cell or by therapeutic intervention.

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Engineering of a Polydisperse Small Heat-Shock Protein Reveals Conserved Motifs of Oligomer Plasticity

Cited by 3 publications

References 69 publications

A weakened interface in the P182L variant of HSP27 associated with severe Charcot‐Marie‐Tooth neuropathy causes aberrant binding to interacting proteins

A weakened interface in the P182L variant of HSP27 associated with severe Charcot‐Marie‐Tooth neuropathy causes aberrant binding to interacting proteins

Dysregulated interactions triggered by a neuropathy-causing mutation in the IPV motif of HSP27

Release of a disordered domain enhances HspB1 chaperone activity toward tau

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