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Multiple sites in ?B-crystallin modulate its interactions with desmin filaments assembled in vitro.

ABSTRACT: The ?3- and ?8-strands and C-terminal residues 155-165 of ?B-crystallin were identified by pin arrays as interaction sites for various client proteins including the intermediate filament protein desmin. Here we present data using 5 well-characterised ?B-crystallin protein constructs with substituted ?3- and ?8-strands and with the C-terminal residues 155-165 deleted to demonstrate the importance of these sequences to the interaction of ?B-crystallin with desmin filaments. We used electron microscopy of negatively stained samples to visualize increased interactions followed by sedimentation assays to quantify our observations. A low-speed sedimentation assay measured the ability of ?B-crystallin to prevent the self-association of desmin filaments. A high-speed sedimentation assay measured ?B-crystallin cosedimentation with desmin filaments. Swapping the ?8-strand of ?B-crystallin or deleting residues 155-165 increased the cosedimentation of ?B-crystallin with desmin filaments, but this coincided with increased filament-filament interactions. In contrast, substitution of the ?3-strand with the equivalent ?A-crystallin sequences improved the ability of ?B-crystallin to prevent desmin filament-filament interactions with no significant change in its cosedimentation properties. These data suggest that all three sequences (?3-strand, ?8-strand and C-terminal residues 155-165) contribute to the interaction of ?B-crystallin with desmin filaments. The data also suggest that the cosedimentation of ?B-crystallin with desmin filaments does not necessarily correlate with preventing desmin filament-filament interactions. This important observation is relevant not only to the formation of the protein aggregates that contain both desmin and ?B-crystallin and typify desmin related myopathies, but also to the interaction of ?B-crystallin with other filamentous protein polymers.

SUBMITTER: Houck SA

PROVIDER: S-EPMC3212511 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Multiple sites in αB-crystallin modulate its interactions with desmin filaments assembled in vitro.

Houck Scott A SA Landsbury Andrew A Clark John I JI Quinlan Roy A RA

PloS one 20111109 11

The β3- and β8-strands and C-terminal residues 155-165 of αB-crystallin were identified by pin arrays as interaction sites for various client proteins including the intermediate filament protein desmin. Here we present data using 5 well-characterised αB-crystallin protein constructs with substituted β3- and β8-strands and with the C-terminal residues 155-165 deleted to demonstrate the importance of these sequences to the interaction of αB-crystallin with desmin filaments. We used electron micros ...[more]

PMID: 22096479

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Project description:Molecular chaperones including the small heat shock proteins, alphaB crystallin and sHSP27 participate in the assembly, disassembly, and reorganization of the cytoskeleton during cell development and differentiation. While alphaB crystallin and sHSP27 stabilize and modulate filament assembly and re-organization, the sequences and structural domains mediating interactions between these proteins and filaments are unknown. It is important to define these interactive domains in order to understand differential interactions between chaperones and stable or unfolding filaments and their function in the cellular stress response. Protein pin arrays identified sequences in human alphaB crystallin that selectively interacted with native or partially unfolded filament proteins desmin, glial-fibrillary acidic protein, and actin. Circular dichroism spectroscopy determined differences in the structure of these filaments at 23 and 45 degrees C. Seven alphaB crystallin sequences had stronger interactions with desmin and six sequences had stronger interactions with glial-fibrillary acidic protein at 23 degrees C than at 45 degrees C. The alphaB crystallin sequences (33)LESDLFPTSTSLSPFYLRPPSFLR(56) and (129)DPLTITSSLSSDGV(145) had the strongest interactions with actin at 23 degrees C, while (57)APSWFDTG(64), (111)HGFISREF(118), (145)VNGPRKQVSG(154), and (155)PERTIPITREEK(165) had the strongest interactions with actin at 45 degrees C. The actin interactive sequences of alphaB crystallin overlapped with previously identified alphaB crystallin chaperone sequences and were synthesized to evaluate their effect on the assembly and aggregation of actin. Full-length alphaB crystallin and the core domain chaperone sequence (131)LTITSSLSSDGV(143) promoted actin polymerization at 37 degrees C and inhibited depolymerization and aggregation at 50 degrees C. The results support the hypothesis that interactive domains in alphaB crystallin have multiple functions in stabilizing the cytoskeleton and protecting cytosolic proteins from unfolding.

Dataset Information

Multiple sites in ?B-crystallin modulate its interactions with desmin filaments assembled in vitro.

Publications

Multiple sites in αB-crystallin modulate its interactions with desmin filaments assembled in vitro.

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