Oxidatively Modified Protein‐Disulfide Isomerase–Associated 3 Promotes Dyskerin Pseudouridine Synthase 1–Mediated Malignancy and Survival of Hepatocellular Carcinoma Cells

Abstract

Dyskerin pseudouridine synthase 1 (DKC1) is a conserved gene encoding the RNA-binding protein dyskerin, which is an essential component of the telomerase holoenzyme. DKC1 up-regulation is frequently observed in many different human cancers including hepatocellular carcinoma (HCC); however, its regulatory mechanisms remain unclear. Thus, we investigated the regulatory mechanism of DKC1 in HCC progression. We found that protein-disulfide isomerase-associated 3 (PDIA3) interacted with the DKC1 regulatory DNA in HCC cells but not in HCC cells with elevated reactive oxygen species (ROS) levels, using liquid chromatographic-tandem mass spectrometric analysis after isolating the DKC1 regulatory region binding proteins. PDIA3 repressed DKC1 expression in HCC cells by recognizing the G-quadruplex DNA at the DKC1 location. However, oxidative modification of PDIA3 induced by ROS redistributed this protein into the cytosolic regions, which stimulated DKC1 expression. We also identified Met338 in PDIA3 as the oxidatively modified residue and validated the effect of oxidative modification using an ectopic expression system, a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 knock-in system, and a xenograft mouse model. We observed that oxidatively modified PDIA3 promoted DKC1-mediated malignancy and survival of HCC cells in vitro and in vivo. HCC tissues showed a positive association with ROS, cytoplasmic PDIA3, and nuclear DKC1 levels. HCC patients with high PDIA3 protein and DKC1 mRNA levels also displayed reduced recurrence-free survival rates. Cumulatively, the results showed that cytoplasmic PDIA3 activity could be essential in raising DKC1 expression in HCC progression and predicting poor prognoses in HCC patients. Conclusion: Our study indicates that the elevated ROS levels in HCC modulate cytoplasmic PDIA3 levels, resulting in HCC cell survival through DKC1 up-regulation.

SERPINA3 is a key modulator of HNRNP-K transcriptional activity against oxidative stress in HCC

“…We previously documented that H 2 O 2 treatment induced a telomere elongation and cell invasion [5,6,12] in HCC cells. In this study, to further examine the molecular linkage between telomere maintenance and cell invasion, we isolated cells with H 2 O 2 -mediated long telomeres (cells with top 6% of telomere intensity) and short telomeres (cells with bottom 66% of telomere intensity), respectively (Fig.…”

“…ROS triggers several signal transduction pathways through the oxidation modification of protein kinases and tyrosine phosphatases, including protein kinase C and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) in tumorigenesis [[19], [20], [21]]. ROS also incrementally affects nuclear localization and the affinity of regulatory DNA-interaction of transcription factors, such as nuclear factor kappa B, p53, activating protein-1, and protein-disulfide isomerase-associated 3 in tumor cell proliferation and survival [12,22,23]. In addition, ROS influences the DNA damage response and repair systems through the phosphorylation of ataxia-telangiectasia mutated protein kinase, p53, and checkpoint kinase 2 [24].…”

A Gallium(III) Complex that Engages Protein Disulfide Isomerase A3 (PDIA3) as an Anticancer Target

“…PDI family has been well known to play important roles in protein folding by helping substrate proteins form correct arrangement of disulfides to reach their natively folded states, and they are primarily anchored in ER in eukaryotic cells. Among the four PDI members,P DIA3 [18] has the highest number of spectra counts and quantifiable peptides.Itisalso found among the proteins whose mRNAwere up-regulated (> 1.5 fold) in the mRNAprofiling.W etherefore decided to take PDIA3 to biochemically and functionally verify its interaction with Ga-1.…”

A Gallium(III) Complex that Engages Protein Disulfide Isomerase A3 (PDIA3) as an Anticancer Target

“…PDI family has been well known to play important roles in protein folding by helping substrate proteins form correct arrangement of disulfides to reach their natively folded states, and they are primarily anchored in ER in eukaryotic cells. Among the four PDI members, PDIA3 [18] has the highest number of spectra counts and quantifiable peptides. It is also found among the proteins whose mRNA were up‐regulated (>1.5 fold) in the mRNA profiling.…”