Transcription regulation of caspase-1 by R393 of HIPPI and its molecular partner HIP-1

Abstract

Earlier we have shown that exogenous expression of HIPPI, a molecular partner of Huntingtin interacting protein HIP-1, induces apoptosis and increases expression of caspases-1, -8 and -10 in HeLa and Neuro2A cells. The C-terminal pseudo death effector domain of HIPPI (pDED-HIPPI) specifically interacts with the putative promoter sequences of these genes. In the present manuscript, we predict from structural modeling of pDED-HIPPI that R393 of HIPPI is important for such interaction. R393E mutation in pDED-HIPPI decreases the interaction with the putative promoter of caspase-1 in cells. Expression of caspase-1 is decreased in cells expressing mutant pDED-HIPPI in comparison to that observed in cells expressing wild type pDED-HIPPI. Using HIP-1 knocked down cells as well as over expressing HIP-1 with mutation at its nuclear localization signal and other deletion mutations, we demonstrate that translocation of HIPPI to the nucleus is mediated by HIP-1 for the increased expression of caspase-1. HIPPI-HIP-1 heterodimer is detected in cytoplasm as well as in the nucleus and is associated with transcription complex in cells. Taking together, we are able to show the importance of R393 of HIPPI and the role of HIPPI-HIP-1 heterodimer in the transcription regulation of caspase-1.

Regulation of RE1 Protein Silencing Transcription Factor (REST) Expression by HIP1 Protein Interactor (HIPPI)

“…Construction of Plasmids-Constructions of GFP-Hippi, GFP-wpDED (coding for amino acids 335-429 of full-length HIPPI) and GFP-mpDED (R393E) clones have been described earlier (21). Construction of wild type huntingtin exon 1 cloned in DsRedC1 vector (DsRed-16Q) was described earlier (23).…”

“…Knockdown of HIP1 and HIPPI by siRNA-Knockdown of HIP1 by gene-specific siRNA in HeLa and Neuro2A cells was described earlier (21).…”

“…Subcellular Fractionation and Western Blot Analysis-Methods for subcellular fractionation and Western blot were essentially the same as described previously (21). Briefly, cells grown in 100-mm Petri dishes were washed with ice-cold PBS and harvested at 300 ϫ g for 3 min at 4°C.…”

“…HIPPI directly interacts in vitro and in vivo with a specific 9-bp DNA sequence, AAA-GACATG, present at the putative promoter of the caspase-1 gene (19,20). The protein lacks any conventional nuclear localization signal (NLS) and is carried to the nucleus by the NLS present at the C terminus of HIP1 (21). The HIPPI-HIP1 heterodimeric complex is then recruited to the promoter of caspase-1 to regulate its transcription (21).…”

“…The protein lacks any conventional nuclear localization signal (NLS) and is carried to the nucleus by the NLS present at the C terminus of HIP1 (21). The HIPPI-HIP1 heterodimeric complex is then recruited to the promoter of caspase-1 to regulate its transcription (21). The emergence of HIPPI as a new transcription regulator for caspase-1 motivated us to investigate the global change in gene expression brought about by HIPPI.…”