Oligomers of Mutant Glial Fibrillary Acidic Protein (GFAP) Inhibit the Proteasome System in Alexander Disease Astrocytes, and the Small Heat Shock Protein αB-Crystallin Reverses the Inhibition

Abstract

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...

Emerging role of autophagy in pediatric neurodegenerative and neurometabolic diseases

“…72 Interestingly, proteasome activity was found to be significantly impaired by mutant GFAP, leading to a disruption of normal protein turnover, a finding that could be partly reversed by expressing α-B-crystallin. 70,73 Importantly, proteasome inhibition in AxD has been linked to the induction of autophagy via mTOR. 74 This is in agreement with the general notion that autophagic degradation is enhanced when the proteasome is dysfunctional or overwhelmed.…”

“…66 Using different mouse models, the accumulation of RFs was shown to be caused by mutant GFAP protein and also by overexpression of wild-type human GFAP, suggesting that elevation of total levels of GFAP is a critical element. 67,68 All GFAP mutations discovered in AxD patients allow production of functional full-length mutant protein, 69 but mutant GFAP tends to form abnormally large, soluble oligomers, 70 and hence, progressive accumulation of mutant GFAP impacts protein metabolism in several ways. Astrocytes and RFs in AxD contain abundant amounts of chaperone proteins suggesting cell stress potentially through the accumulation of misfolded GFAP.…”

HspB5/αB-Crystallin in the Brain

“…Recent work has shown that GFAP inhibits the proteasome and that this effect is increased in mutated GFAP leading to less proteasomal degradation of GFAP. Interestingly, HspB5 reverses this inhibitory effect of mutated GFAP on the proteasome which might be one mechanism of how HspB5 exerts its cytoprotective effect in Alexander disease (Tang et al 2010 ). (Head et al 1993 ;Iwaki et al 1989Iwaki et al , 1993 In vitro…”

Lopinavir Up-Regulates Expression of the Antiviral Protein Ribonuclease L in Human Papillomavirus-Positive Cervical Carcinoma Cells

“…Indeed, as we have discussed, lopinavir induced changes in the levels of several other apoptosis-related proteins and it is also significant that the majority of the proteins listed in Table 1 are known to be regulated by proteasomal degradation. On this point, an interesting finding was the observed lopinavir-induced down-regulation of GFAP which is known to be capable of inhibiting the proteasome [25]. Since p53 is a target of high-risk HPV E6, we searched the Virus Molecular INTeraction (VirusMINT) database [26] for any other known E6 and E7 targeted proteins which are affected by lopinavir treatment (Table 1).…”