HDACis were found to increase the quantity of cystic fibrosis transmembrane conductance regulator by reducing its degradation (29). gene encoding the lysosomal enzyme glucocerebrosidase (GCase), leading to accumulation of toxic amounts of glucocerebroside and subsequent organ and metabolic dysfunction. Approximately 360 unique mutations have been identified in GD, most of them missense mutations (1, 2). Our previous study revealed that these missense mutations result Ipfencarbazone in a reduction of protein stability, rather than disruption of intrinsic Ipfencarbazone enzymatic activity (3, 4). GCase undergoes significant posttranslational modification in the endoplasmic reticulum (ER). Nascent peptides form transient protein complexes with chaperone and cochaperone proteins, which facilitate proper folding and modification (5). Missense mutations in GCase destabilize the protein by introducing an unnatural conformation that results in altered chaperone binding, rendering the peptide vulnerable to recognition by E3 ligases (parkin and c-cbl) and proteasome-associated degradation (3, 6). Identifying key chaperone proteins that determine GCase proteostasis is usually potentially of great importance in targeting treatment of patients with GD. Histone deacetylase inhibitors (HDACis) are a class of compounds first found to interfere with histone acetylation. HDACis such as valproic acid have been used to treat psychiatric/neurologic disorders, inflammatory diseases, and cancers (7C9). Along with their histone-modifying effects, HDACis translocate from the cell nucleus to the cytoplasm and are involved in posttranslational modification of nonhistone and cytoplasmic proteins (10, 11). Indeed, HDACis have been shown to remove acetyl moieties from heat shock protein (Hsp) 70, Hsp90, and tubulin (12C15). Several recent discoveries suggest that HDACis are effective in treating inherited diseases that arise from misfolding of proteins, such as GD, cystic fibrosis, Huntington disease, and type C NiemannCPick disease (16C19). The molecular mechanism of how HDACis affect proteostasis remains unclear, however. In the present study, we investigated key molecular chaperones that mediate GCase degradation. Using two common mutations for type I (N370S/N370S) and Ipfencarbazone Rabbit polyclonal to ACSF3 type II/III (L444P/L444P) GD, we discovered that misfolding of GCase results in fundamental changes in the protein expression profile of Ipfencarbazone ER stress/ER-associated degradation (ERAD)-related genes as well as molecular chaperones. Among these chaperones, Hsp90 is essential for the degradation of misfolded GCase. Hsp90 recognizes misfolded GCase and guides the nascent protein through a valosin-containing protein (VCP)-associated degradation pathway (20, 21). HDACis cause hyperacetylation of the middle domain name of Hsp90, resulting in limited recognition of GCase mutants by Hsp90 and increased levels of GCase. Results Abnormal Degradation and ER Retention of Mutants. In patients with GD, nascent GCase peptides bearing different pathogenic mutations acquire unnatural conformations and are not folded into the appropriate tertiary structure. We first investigated the subcellular distribution of GCase mutants in fibroblasts derived from either type I (N370S) or type II (L444P) GD. Consistent with previous findings, we confirmed a fundamental loss of GCase in patient-derived fibroblasts. In addition, GCase from patients with GD was consistently restricted to the ER, implying that GCase cannot be targeted to the correct subcellular compartment for assembly and function. In contrast to this, in normal fibroblasts GCase was successfully exported from ER, suggesting correct protein folding and translocation (Fig. 1increased GCase over a 2-d period. Inhibition on resulted in decreased protein levels. (or increased GCase enzyme activity in fibroblasts derived from patients with GD. Inhibition of further decreased GCase activity. (or increased the quantity of mutant GCases, whereas inhibition on reduced the quantity of GCase protein (Fig. 1mutants (N370S). We used the same cell line expressing WT GBA as a baseline. We identified a global increase in chaperonin/cochaperonin gene expression in N370S cells compared with WT. These include critical protein folding machinery genes, such as (Fig. 2mutants in HeLa cells, combined with WT (Hsp90-WT) or dominant-negative Hsp90 recombinant (Hsp90-D88N). Consistent with previous findings, we identified abnormally Ipfencarbazone increased ubiquitination of GCase mutants. Cotransfection of Hsp90-WT resulted in similar trends in ubiquitination, indicating that endogenous Hsp90 is usually.