Aggregate-Prone R120GCRYAB Triggers Multifaceted Modifications of the Thioredoxin System

Abstract

Aims: The human mutation R120G in the aB-crystallin (CRYAB) causes a multisystemic disease that is characterized by hypertrophic cardiomyopathy and cytoplasmic protein aggregates. In transgenic mice, human R120GCRYAB (hR120GTg) expression in heart sequentially modifies the REDOX status, in part by the activation of the nuclear factor, erythroid derived 2, like 2 (Nrf2). Thioredoxin system (TS) components are NRF2 target genes, so it could be hypothesized that TS was affected in hR120GTg mice. Results: Transgenic hearts overexpressed thioredoxin 1 (Trx1), which was identified by isotope coded affinity tag-mass spectrometry, among hundreds of peptides displaying an increased reduced/oxidized ratio. Coupled to this higher level of reduced cysteines, the activity of thioredoxin reductase 1 (TrxR1) was augmented by 2.5-fold. Combining mutiple experimental approaches, the enzymatic regulation of TrxR1 by a histone deacetylase 3 (HDAC3)-dependent level of acetylation was confirmed. In vitro and in vivo functional tests established that TrxR1 activity is required to mitigate aggregate development, and this could be mediated by Bcl-2-associated athanogene 3 (BAG3) as a potential TS substrate. Innovation and Conclusions: This study uncovers the compartmentalized changes and the involvement of TS in the cardiac stress response elicited by misfolded proteins such as R120GCRYAB. Our work suggests that R120GCRYAB triggers a defensive pathway acting through the newly identified interacting partners HDAC3, TrxR1, and BAG3 to counter aggregate growth. Therefore, those interactors may function as modifier genes contributing to the variable onset and expressivity of such human diseases. Furthermore, our work underscores the potential organismal effects of pharmacological interventions targeting TS and HDAC.

Modulation of signaling mechanisms in the heart by thioredoxin 1

“…In these mice, both reduced Trx1 and TrxR1 are upregulated through Nrf2 activation and prevent accumulation of cytoplasmic protein aggregates. This finding is of interest because it has been suggested that protein aggregates are formed due to reductive stress in desmin-related myopathy [66]. The fact that an antioxidant mechanism can reduce protein aggregates even in the presence of reductive stress conditions indicates that the protective effect of the Trx1 system may be compartmentalized.…”

MDR1 mediated chemoresistance: BMI1 and TIP60 in action

“…RNA was first retro-transcribed using iScript cDNA Synthesis kit (Bio-Rad, Hercules, CA), and then either real-time PCR was carried out using iTaq SYBR Green Master Mix (Bio-Rad). The comparative C t (2 −ΔΔ C T ) method [74] was used to calculate the relative abundance of the mRNA and compared with that of housekeeping gene expression 18S rRNA as previously described [75]. For end point PCR, RNA was isolated, retro-transcribed into cDNA and then amplified using 2× Taq polemerase (NEB) and primers.…”

“…Immunoprecipitation was performed using with Agarose A/G beads (SCBT-2003), and cell lystaes or immunoprecipitates were separated by SDS-PAGE and Western immunoblotting analysis was performed using standard protocol as described previously [75]. When required, DNASE1 (Sigma) digestion was done during immunoprecipitation with 150U and 250U DNASE1.…”

Mitochondrial BMI1 maintains bioenergetic homeostasis in cells

“…Cell lysis, immunoprecipitation (IP), and immunoblotting were performed and quantified as described previously (16).…”