The Cardiomyopathy and Lens Cataract Mutation in αB-crystallin Alters Its Protein Structure, Chaperone Activity, and Interaction with Intermediate Filaments in Vitro

Perng, Ming Der; Muchowski, Paul J.; IJssel, Paul van den; Wu, Gabrielle J. S.; Hutcheson, Aileen M.; Clark, John I.; Quinlan, Roy A.

doi:10.1074/jbc.274.47.33235

Cited by 188 publications

(168 citation statements)

References 59 publications

(73 reference statements)

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“…First, in pulse-chase experiments, newly synthesized ezrin incorporated in Triton-insoluble complexes with keratins very rapidly (Ͻ10 min) and transiently ( Figure 6B). It is recognized, however, that coimmunoprecipitation of newly synthesized ezrin with IFs might be mediated by other proteins such as, for example, IF-associated chaperones (Perng et al, 1999). The second line of evidence showed that dormant, but not active, ezrin can bind directly to keratins in vitro ( Figure 7, B and C).…”

Section: Discussionmentioning

confidence: 96%

Intermediate Filaments Interact with Dormant Ezrin in Intestinal Epithelial Cells

Wald

Oriolo

Casanova

et al. 2005

MBoC

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Ezrin connects the apical F-actin scaffold to membrane proteins in the apical brush border of intestinal epithelial cells. Yet, the mechanisms that recruit ezrin to the apical domain remain obscure. Using stable CACO-2 transfectants expressing keratin 8 (K8) antisense RNA under a tetracycline-responsive element, we showed that the actin-ezrin scaffold cannot assemble in the absence of intermediate filaments (IFs). Overexpression of ezrin partially rescued this phenotype. Overexpression of K8 in mice also disrupted the assembly of the brush border, but ezrin distributed away from the apical membrane in spots along supernumerary IFs. In cytochalasin D-treated cells ezrin localized to a subapical compartment and coimmunoprecipitated with IFs. Overexpression of ezrin in undifferentiated cells showed a Triton-insoluble ezrin compartment negative for phospho-T567 (dormant) ezrin visualized as spots along IFs. Pulse-chase analysis showed that Triton-insoluble, newly synthesized ezrin transiently coimmunoprecipitates with IFs during the first 30 min of the chase. Dormant, but not active (p-T567), ezrin bound in vitro to isolated denatured keratins in Far-Western analysis and to native IFs in pull-down assays. We conclude that a transient association to IFs is an early step in the polarized assembly of apical ezrin in intestinal epithelial cells. INTRODUCTIONEzrin, a member of the ezrin-radixin-moesin (ERM) family, is a conspicuous component of the apical F-actin-based scaffold in simple epithelial cells. It connects NHERF/EBP-50 (which in turn binds important membrane proteins such as CFTR and NHE-3) to F-actin and also enables protein kinase A-mediated regulation of apical channels (Kurashima et al., 1999;Louvet-Vallée, 2000;Weinman et al., 2000Weinman et al., , 2001. Because ezrin is the earliest protein of this complex scaffold to be recruited, it has long been considered as an organizer of the brush border (Bretscher et al., 1997). In support of that view, ezrin knockout mice show a distinctive brush-border phenotype with much shorter microvilli that resemble undifferentiated stages (Saotome et al., 2004). Furthermore, expression of ezrin in fibroblasts is sufficient to organize microvilli (Shaw et al., 1998). After translation, ezrin initially adopts a "dormant" configuration, in which the C-and N-terminal domains bind to each other. On phosphorylation in T567, it changes to an open "active" configuration, freeing the C-terminal domain (C-ERMAND) to bind F-actin and the N-terminal domain (N-ERMAND) to bind NHERF or transmembrane proteins (Bretscher et al., 2000). Although the mechanism of activation and assembly of ezrin has been studied in great detail by several groups, the mechanisms that ensure that ezrin assembly occur mostly under the apical membrane of simple epithelial cells remain obscure. Fievet et al. (2004) have shown that binding to phosphatidylinositol 4,5-diphosphate (PIP 2 ) is a prerequisite for activation. However, PIP 2 is not known to be apically polarized in epithelial cells, rather it is basol...

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Section: Discussionmentioning

confidence: 96%

Intermediate Filaments Interact with Dormant Ezrin in Intestinal Epithelial Cells

Wald

Oriolo

Casanova

et al. 2005

MBoC

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“…72 An in vitro chaperone assay demonstrated that the mutant p.Arg120Gly alphaB-crystallin becomes functionally deficient. 89 Expression of the mutant alphaB-crystallin in SW13 and BHK21 cells leads to formation of abnormal aggregates that contain both desmin and alphaB-crystallin. 8 Transgenic mice expressing mutant alphaB crystallin show the presence of abnormal desmin and alphaB-crystallin aggregates in the cardiomyocytes; formation of aggregates is the result of the loss of protection from alphaBcrystallin.…”

Section: Role Of Alphab-crystallinmentioning

confidence: 99%

Intermediate Filament Diseases: Desminopathy

Goldfarb

Olivé

Vicart³

et al. 2008

Advances in Experimental Medicine and Biology

107

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Desminopathy is one of the most common intermediate filament human disorders associated with mutations in closely interacting proteins, desmin and alphaB-crystallin. The inheritance pattern in familial desminopathy is characterized as autosomal dominant or autosomal recessive, but many cases have no family history. At least some and likely most sporadic desminopathy cases are associated with de novo DES mutations. The age of disease onset and rate of progression may vary depending on the type of inheritance and location of the causative mutation. Typically, the illness presents with lower and later upper limb muscle weakness slowly spreading to involve truncal, neckflexor, facial and bulbar muscles. Skeletal myopathy is often combined with cardiomyopathy manifested by conduction blocks, arrhythmias and chronic heart failure resulting in premature sudden death. Respiratory muscle weakness is a major complication in some patients. Sections of the affected skeletal and cardiac muscles show abnormal fibre areas containing chimeric aggregates consisting of desmin and other cytoskeletal proteins. Various DES gene mutations: point mutations, an insertion, small in-frame deletions and a larger exon-skipping deletion, have been identified in desminopathy patients. The majority of these mutations are located in conserved alpha-helical segments, but additional mutations have recently been identified in the tail domain. Filament and network assembly studies indicate that most but not all disease-causing mutations make desmin assembly-incompetent and able to disrupt a pre-existing filamentous network in dominant-negative fashion. AlphaBcrystallin serves as a chaperone for desmin preventing its aggregation under various forms of stress; mutant CRYAB causes cardiac and skeletal myopathies identical to those resulting from DES mutations.

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“…The wild type ␣B-crystallin, K174X and A171X proteins were prepared essentially as described (29), but adding polyethyleneimine (final concentration 0.1% (w/v)) to remove contaminating DNA (10,30) before purification by ion exchange chromatography on a Fractogel TMAE column. Peak fractions were pooled and then dialyzed into 20 mM Tris-HCl, 100 mM NaCl, pH 7.4, in preparation for size exclusion chromatography on a Fractogel EMD BioSEC Superformance column (60 ϫ 1.6 cm; VWR, UK).…”

Section: Protein Expression and Purification Of ␣B-crystallin Mutantsmentioning

confidence: 99%

Truncation of αB-Crystallin by the Myopathy-causing Q151X Mutation Significantly Destabilizes the Protein Leading to Aggregate Formation in Transfected Cells

Hayes

Devlin

Quinlan

2008

Journal of Biological Chemistry

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Here we investigate the effects of a myopathy-causing mutation in ␣B-crystallin, Q151X, upon its structure and function. This mutation removes the C-terminal domain of ␣B-crystallin, which is expected to compromise both its oligomerization and chaperone activity. We compared this to two other ␣B-crystallin mutants (450delA, 464delCT) and also to a series of C-terminal truncations (E164X, E165X, K174X, and A171X). We find that the effects of the Q151X mutation were not always as predicted. Specifically, we have found that although the Q151X mutation decreased oligomerization of ␣B-crystallin and even increased some chaperone activities, it also significantly destabilized ␣B-crystallin causing it to self-aggregate. This conclusion was supported by our analyses of both the other disease-causing mutants and the series of C-terminal truncation constructs of ␣B-crystallin. The 450delA and 464delCT mutants could only be refolded and assayed as a complex with wild type ␣B-crystallin, which was not the case for Q151X ␣B-crystallin. From these studies, we conclude that all three disease-causing mutations (450delA, 464delCT, and Q151X) in the C-terminal extension destabilize ␣B-crystallin and increase its tendency to self-aggregate. We propose that it is this, rather than a catastrophic loss of chaperone activity, which is a major factor in the development of the reported diseases for the three disease-causing mutations studied here. In support of this hypothesis, we show that Q151X ␣B-crystallin is found mainly in the insoluble fraction of cell extracts from transient transfected cells, due to the formation of cytoplasmic aggregates.The crystallins were first identified as the major proteins of the eye lens and their subsequent classification into ␣-, ␤-, and ␥-crystallins (1) allowed different functional properties to be assigned to the ␣-and ␤␥-crystallin groups (2). The ␣-crystallins are two distinct proteins derived from two separate genes, ␣A-and ␣B-crystallin (2). Whereas ␣A-crystallin is almost entirely lens specific (3), ␣B-crystallin is expressed widely in other tissues, most notably astrocytes (4) and muscle (5). In the early 1980s, pioneering work from Klemenz and co-workers (6) and Horwitz (7) laboratories established that ␣B-crystallin was a protein chaperone and a member of the small heat shock protein (sHSP) 2 family, that is now known to comprise 10 different human proteins (8).When the first mutation (R120G) in ␣B-crystallin was reported, it was found to cause both cataract and desmin-related myopathy in the affected patients (9). Characteristic histopathological aggregates of the muscle intermediate filament protein desmin were observed (9), strongly suggesting that there is an important functional interaction between intermediate filaments and ␣B-crystallin. Subsequent analyses showed that the R120G ␣B-crystallin mutation induced increased binding for desmin intermediate filaments (10), providing an explanation for the formation of the desmin aggregates in the muscles of the affected individuals. Coinci...

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The Cardiomyopathy and Lens Cataract Mutation in αB-crystallin Alters Its Protein Structure, Chaperone Activity, and Interaction with Intermediate Filaments in Vitro

Cited by 188 publications

References 59 publications

Intermediate Filaments Interact with Dormant Ezrin in Intestinal Epithelial Cells

Intermediate Filaments Interact with Dormant Ezrin in Intestinal Epithelial Cells

Intermediate Filament Diseases: Desminopathy

Truncation of αB-Crystallin by the Myopathy-causing Q151X Mutation Significantly Destabilizes the Protein Leading to Aggregate Formation in Transfected Cells

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