Seven members of the small heat shock protein (sHSP) family are exceptional with respect to their constitutive high abundance in muscle tissue. It has been suggested that sHSPs displaying chaperone-like properties may stabilize myofibrillar proteins during stress conditions and prevent them from loss of function. In the present study five sHSPs (alphaB-crystallin, MKBP, HSP25, HSP20, and cvHSP) were investigated with respect to similarities and differences of their expression in heart and skeletal muscle under normal and ischemic conditions. In ischemic heart and skeletal muscle these five sHSPs translocated from cytosol to the Z-/I-area of myofibrils. Myofibrillar binding of all sHSPs was very tight and resisted for the most part extraction with 1 M NaSCN or 1 M urea. MKBP and HSP20 became extracted by 1 M NaSCN to a significant extent indicating that these two sHSPs may bind partially to actin-associated proteins which were completely extracted by this treatment. Ultrastructural localization of alphaB-crystallin showed diffuse distribution of immunogold label throughout the entire I-band in skeletal muscle fibers whereas in cardiomyocytes alphaB-crystallin was preferentially located at the N-line position of the I-band. These observations indicate different myofibrillar binding sites of alphaB-crystallin in cardiomyocytes versus skeletal muscle fibers. Further differences of the properties of sHSPs could be observed regarding fiber type distribution of sHSPs. Thus sHSPs form a complex stress-response system in striated muscle tissue with some common as well as some distinct functions in different muscle types.

. (1999) 'The cardiomyopathy and lens cataract mutation in B-crystallin alters its protein structure, chaperone activity, and interaction withintermediate laments in vitro.', Journal of biological chemistry., 274 (47). pp. 33235-33243. Further information on publisher's website: Use policyThe full-text may be used and/or reproduced, and given to third parties in any format or medium, without prior permission or charge, for personal research or study, educational, or not-for-prot purposes provided that:• a full bibliographic reference is made to the original source • a link is made to the metadata record in DRO • the full-text is not changed in any way The full-text must not be sold in any format or medium without the formal permission of the copyright holders.Please consult the full DRO policy for further details.

Here, we describe the early events in the disease pathogenesis of Alexander disease. This is a rare and usually fatal neurodegenerative disorder whose pathological hallmark is the abundance of protein aggregates in astrocytes. These aggregates, termed "Rosenthal fibers," contain the protein chaperones alpha B-crystallin and HSP27 as well as glial fibrillary acidic protein (GFAP), an intermediate filament (IF) protein found almost exclusively in astrocytes. Heterozygous, missense GFAP mutations that usually arise spontaneously during spermatogenesis have recently been found in the majority of patients with Alexander disease. In this study, we show that one of the more frequently observed mutations, R416W, significantly perturbs in vitro filament assembly. The filamentous structures formed resemble assembly intermediates but aggregate more strongly. Consistent with the heterozygosity of the mutation, this effect is dominant over wild-type GFAP in coassembly experiments. Transient transfection studies demonstrate that R416W GFAP induces the formation of GFAP-containing cytoplasmic aggregates in a wide range of different cell types, including astrocytes. The aggregates have several important features in common with Rosenthal fibers, including the association of alpha B-crystallin and HSP27. This association occurs simultaneously with the formation of protein aggregates containing R416W GFAP and is also specific, since HSP70 does not partition with them. Monoclonal antibodies specific for R416W GFAP reveal, for the first time for any IF-based disease, the presence of the mutant protein in the characteristic histopathological feature of the disease, namely Rosenthal fibers. Collectively, these data confirm that the effects of the R416W GFAP are dominant, changing the assembly process in a way that encourages aberrant filament-filament interactions that then lead to protein aggregation and chaperone sequestration as early events in Alexander disease.

The R120G mutation in ␣B-crystallin causes desmin-related myopathy. There have been a number of mechanisms proposed to explain the disease process, from altered protein processing to loss of chaperone function. Here, we show that the mutation alters the in vitro binding characteristics of ␣B-crystallin for desmin filaments. The apparent dissociation constant of R120G ␣B-crystallin was decreased while the binding capacity was increased significantly and as a result, desmin filaments aggregated. These data suggest that the characteristic desmin aggregates seen as part of the disease histopathology can be caused by a direct, but altered interaction of R120G ␣B-crystallin with desmin filaments. Transfection studies show that desmin networks in different cell backgrounds are not equally affected. Desmin networks are most vulnerable when they are being made de novo and not when they are already established. Our data also clearly demonstrate the beneficial role of wild-type ␣B-crystallin in the formation of desmin filament networks. Collectively, our data suggest that R120G ␣B-crystallin directly promotes desmin filament aggregation, although this gain of a function can be repressed by some cell situations. Such circumstances in muscle could explain the late onset characteristic of the myopathies caused by mutations in ␣B-crystallin. INTRODUCTIONThe study of many different human diseases caused by mutations in intermediate filament (IF) proteins (McLean and Lane, 1995;Fuchs and Cleveland, 1998;Coulombe and Omary, 2002; has shown that filament aggregation is a common feature (van den IJssel et al., 1999), implying that 10-nm filaments need to be arranged in networks to be functional. What then determines the distribution and location of IF networks in cells? IFs are dynamic structures, individually and collectively (Helfand et al., 2003). The composition of the IFs themselves is clearly a very important factor because that can influence the distribution of filaments (Cary and Klymkowsky, 1994a,b) and their dynamics (Chou et al., 2003) and the effect of some intermediate filament mutations (Zhou et al., 2003). The filament composition can also determine the spacing between filaments, an important part of organizing individual filaments into an ordered arrangement or network. So vimentin, but not nestin, facilitates the correct spacing of glial fibrillary acidic protein filaments (Eliasson et al., 1999) and, of course, altering the proportion of the different neurofilament proteins changes the packing density of neurofilaments (Xu et al., 1996). Then, the provision of appropriate docking sites, for instance on the plasma membrane (Borradori and Sonnenberg, 1999;Green and Gaudry, 2000;Garrod et al., 2002), as provided by members of the spectraplakin protein family (Leung et al., 2001;Roper and Brown, 2003) and also by some IF proteins themselves, such as syncoilin (Poon et al., 2002). IF composition and available attachment sites are therefore important factors in network formation.This point has been firmly made for des...

The accumulation of the intermediate filament protein, glial fibrillary acidic protein (GFAP), in astrocytes of Alexander disease (AxD) impairs proteasome function in astrocytes. We have explored the molecular mechanism that underlies the proteasome inhibition. We find that both assembled and unassembled wild type (wt) and R239C mutant GFAP protein interacts with the 20 S proteasome complex and that the R239C AxD mutation does not interfere with this interaction. However, the R239C GFAP accumulates to higher levels and forms more protein aggregates than wt protein. These aggregates bind components of the ubiquitin-proteasome system and, thus, may deplete the cytosolic stores of these proteins. We also find that the R239C GFAP has a greater inhibitory effect on proteasome system than wt GFAP. Using a ubiquitin-independent degradation assay in vitro, we observed that the proteasome cannot efficiently degrade unassembled R239C GFAP, and the interaction of R239C GFAP with proteasomes actually inhibits proteasomal protease activity. The small heat shock protein, ␣B-crystallin, which accumulates massively in AxD astrocytes, reverses the inhibitory effects of R239C GFAP on proteasome activity and promotes degradation of the mutant GFAP, apparently by shifting the size of the mutant protein from larger oligomers to smaller oligomers and monomers. These observations suggest that oligomeric forms of GFAP are particularly effective at inhibiting proteasome activity.Alexander disease (AxD) 2 is a rare but fatal disease of the central nervous system characterized by the presence in astrocytes of Rosenthal fibers, cytoplasmic protein aggregates that contain the intermediate filament protein, glial fibrillary acidic protein (GFAP), ubiquitinated proteins, and small heat shock proteins (shsps) (1). Because of the loss of myelin and oligodendrocytes and a variable loss of neurons, AxD has been historically thought of as a leukodystrophy or neurodegenerative disorder. AxD is, however, a disease of astrocyte dysfunction, as mutations in GFAP are found in the large majority of AxD patients (2). All mutations detected so far are heterozygous coding mutations, and most cases of AxD disease arise through de novo, dominant, GFAP mutations (2). Most of the AxD mutations reside in the 1A, 2A, and 2B segments of the conserved central rod domain of GFAP, but several are located in the tail region (2). Although GFAP mutations must underlie the pathogenesis of AxD, the mechanisms by which GFAP mutations are toxic have not been fully elucidated.The Arg-239 residue is a "hot spot" for GFAP mutagenesis, as substitutions at this arginine are the most frequent mutations found in AxD and appear among different ethnic groups (2). Our previous work has concentrated on the most common substitution, R239C, and has demonstrated that overexpressing GFAP, either wild type (wt) or the R239C mutant, leads to the formation of intracellular protein aggregates (3-5). GFAP accumulation also inhibited proteasome protease activity and increased levels of ubiquit...