Desmin Aggregate Formation by R120G αB-Crystallin Is Caused by Altered Filament Interactions and Is Dependent upon Network Status in Cells

Abstract

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...

Severe muscle disease-causing desmin mutations interfere with in vitro filament assembly at distinct stages

“…In vivo, stability and dynamics of extended filament networks is likely to involve additional proteins, such as IF-associated proteins and chaperones (ref. 26 and references therein), or crossbridging proteins, including plectin and synemin (27,28). Nevertheless, the principal interactions for assembly are well represented by our in vitro experiments (see ref.…”

Chaperone Activity of Small Heat Shock Proteins Underlies Therapeutic Efficacy in Experimental Autoimmune Encephalomyelitis

“…2E). These results were surprising based on the knowledge that chaperone activity of the mutant was less than the parent sequence (19,20) and confirmed in the insulin chaperone assay (Fig. 1E).…”

“…A naturally occurring mutation of this arginine to a glycine compromises the structural integrity of the groove and the resultant chaperone activity of the protein (18). Most importantly, the mutation is physiologically relevant, and is linked to a human desmin myopathy (19,20). The solution of the crystal structure of the mutant revealed a collapse of the groove between the subunits, but otherwise the crystallin domains were intact along with several pockets speculated to be ligand binding sites (21).…”

Chaperones: needed for both the good times and the bad times

“…[61,62]; major histocompatibility complex formation [63,64]; T-cell maturation [65] and antigen presentation [66,67], as well as the formation of muscle sarcomeres [68,69], which includes the assembly of actin [70], desmin [71,72], myosin [73] and titin [74]. Chaperone-assisted assembly of multi-component complexes sometimes uses membrane surfaces [75][76][77] demonstrating that their attentions are not restricted to proteins, but include other biomolecules including DNA [78], RNA [79] and lipids [80][81][82].…”

“…association of sHSPs influence subsequent filament -filament interactions, and is a key factor in the pathology associated with desmin-related myopathy [72]. figure 2).…”