Promotion of a synthetic degradation of activated STAT6 by PARP-1 inhibition: roles of poly(ADP-ribosyl)ation, calpains and autophagy

Abstract

Background We reported that PARP-1 regulates genes whose products are crucial for asthma, in part, by controlling STAT6 integrity speculatively through a calpain-dependent mechanism. We wished to decipher the PARP-1/STAT6 relationship in the context of intracellular trafficking and promoter occupancy of the transcription factor on target genes, its integrity in the presence of calpains, and its connection to autophagy. Methods This study was conducted using primary splenocytes or fibroblasts derived from wild-type or PARP-1−/− mice and Jurkat T cells to mimic Th2 inflammation. Results We show that the role for PARP-1 in expression of IL-4-induced genes (e.g. gata-3) in splenocytes did not involve effects on STAT6 phosphorylation or its subcellular trafficking, rather, it influenced its occupancy of gata-3 proximal and distal promoters in the early stages of IL-4 stimulation. At later stages, PARP-1 was crucial for STAT6 integrity as its inhibition, pharmacologically or by gene knockout, compromised the fate of the transcription factor. Calpain-1 appeared to preferentially degrade JAK-phosphorylated-STAT6, which was blocked by calpastatin-mediated inhibition or by genetic knockout in mouse fibroblasts. The STAT6/PARP-1 relationship entailed physical interaction and modification by poly(ADP-ribosyl)ation independently of double-strand-DNA breaks. Poly(ADP-ribosyl)ation protected phosphorylated-STAT6 against calpain-1-mediated degradation. Additionally, our results show that STAT6 is a bonafide substrate for chaperone-mediated autophagy in a selective and calpain-dependent manner in the human Jurkat cell-line. The effects were partially blocked by IL-4 treatment and PARP-1 inhibition. Conclusions The results demonstrate that poly(ADP-ribosyl)ation plays a critical role in protecting activated STAT6 during Th2 inflammation, which may be synthetically targeted for degradation by inhibiting PARP-1.

Unconventional activation of PRKDC by TNF-α: deciphering its crucial role in Th1-mediated inflammation beyond DNA repair as part of the DNA-PK complex

“…Based on the findings of this study and our previous reports, we propose the new concept of “dispensability of therapeutic targets”. This basically means that inhibiting or reducing most of the target either by enzymatic inhibition or by targeting its integrity by promoting its degradation through proteolysis targeting chimeric (PROTAC) technology [ 37 ] or synthetic degradation [ 41 ] would not affect the normal function of the enzyme, and thus, side effects may not ensue. It is also important to note that in these situations, complete inhibition or elimination of the target is not necessary, as the role of these enzymes in inflammation is highly sensitive to an inhibition of its enzymatic activity or its protein levels.…”