Rabbit Anti-Human DNAJB1 Polyclonal antibody

Alpha-synuclein (alpha-Syn) can misfold and aggregate, causing the degeneration of dopaminergic neurons, as seen in Parkinson's disease (PD). We recently demonstrated that DNAJB6, a co-chaperone found in Lewy bodies (LB), suppresses the aggregation of alpha-Syn in cells and in vitro. In this study, we compared the capacities of DNAJB1 and DNAJB6 to suppress the seeded alpha-Syn aggregation in HEK293 cells expressing alpha-Syn tagged with cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP). The aggregation of alpha-Syn was seeded by the transfection of the cells with recombinant alpha-Syn pre-formed fibrils (PFFs), following the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9-mediated knockout (KO) of these two genes, respectively. We quantified the alpha-Syn aggregation by fluorescence microscopy and fluorescence resonance energy transfer (FRET) analysis. We detected significantly more aggregates in the DNAJB6 KO cells compared with the parental cells, whereas the DNAJB1 KO had no effect on the alpha-Syn aggregation. This is the first evidence that DNAJB6 can suppress alpha-Syn aggregation, induced by exogenous alpha-Syn seeds, in cells. Next, we explored whether this mechanism could be dependent on protein degradation pathways. We observed that the increase in the alpha-Syn PFF-induced aggregation in the DNAJB6 KO cells compared with the parental cells was strongly diminished upon the incubation of the cells with the proteasomal inhibitor MG132. These results consolidate that DNAJB6 is a suppressor of alpha-Syn aggregation, and suggest that DNAJB6 may target misfolded and/or aggregated alpha-Syn for proteasomal degradation.

Fibrolamellar carcinomas are characterized by activation of protein kinase A, a kinase composed of catalytic and regulatory subunits. PRKACA encodes a catalytic subunit of protein kinase A, and almost all fibrolamellar carcinomas have a heterozygous 400-kb deletion that leads to the fusion of DNAJB1 and PRKACA. The resulting DNAJB1-PRKACA fusion transcript is believed to activate protein kinase A by dysregulation of the catalytic portion of the protein. In contrast, PRKAR1A encodes one of the regulatory subunits of protein kinase A. We hypothesized that loss of function of this regulatory unit could also lead to protein kinase A activation and thus to fibrolamellar carcinoma. Because PRKAR1A mutations underlie the Carney complex, we searched for liver tumors in individuals with the Carney complex. We identified 3 individuals with fibrolamellar carcinomas and a personal history of the Carney complex. All three tumors displayed the typical morphology of fibrolamellar carcinoma and were positive for arginase, cytokeratin 7, and cluster of differentiation 68. Fluorescence in situ hybridization was negative for PRKACA rearrangements. However, PRKAR1A sequencing identified pathogenic mutations in two of two cases with successful sequencing. In addition, all three cases were negative for PRKAR1A protein expression, consistent with inactivation of this key regulatory unit of protein kinase A. We also identified one additional fibrolamellar carcinoma in an individual without a documented history of the Carney complex who was negative for PRKACA rearrangements but had loss of PRKAR1A protein expression as well as PRKAR1A mutations. Conclusion: Fibrolamellar carcinoma can be part of the Carney complex; in this setting, fibrolamellar carcinomas have inactivating PRKAR1A mutations instead of the DNAJB1-PRKACA fusion gene found in sporadic fibrolamellar carcinomas, providing an alternate means for activation of protein kinase A. (Hepatology 2017).