Mechanisms of Microbial DNA Sensing by the AIM2 Inflammasome and Structural Study of Polynucleotydyl Transferase1 (pnt1) from Arabidopsis Thaliana

AIM2 (absence in melanoma 2) can sense foreign cytosolic double-stranded DNA (dsDNA) through the dsDNA binding HIN-200 domain at the C-terminus. Once dsDNA is bound to HIN-200 domain, AIM2 can activate the AIM2 inflammasome, resulting in maturation of pro-interleukin-1? by activation of caspase-1. To investigate the mechanism of DNA binding, HIN-200 domain of mouse AIM2 bound to 15bp, 18bp, 20bp and 30bp dsDNAs were purified and crystallized. Diffraction data for four different crystals were collected to about 4? resolution. Interestingly, all the crystals were in the same cubic space group I23 or I213 with almost same unit-cell parameters. Mutagenesis of HIN-200 domain of mouse AIM2 and DNA binding studies revealed amino acid residues involved in DNA binding. These residues were compared with dsDNA binding residues identified from the structure of HIN-200 domains of human AIM2, which suggested mouse AIM2 uses a similar dsDNA binding surface as human AIM2. Polynucleotidyl transferase (PNT1) was discovered in the proteomics study of Argonaute10 (AGO10). AGO10 was known as a key regulator of shoot apical meristem (SAM) maintenance in Arabidopsis. A recent study reported that AGO10 can regulate the developmental processes in SAM by repressing two miRNAs, miR166/165, and bind to these two miRNAs. Sequence analysis of PNT1 showed that it belongs to DnaQ-like 3'-5' exonuclease family, and hypothesized to be involved in RNA regulation with AGO10. PNT1 was purified and strong 3?-5? exonuclease activity was revealed by activity test. Purified protein was crystallized, and the solved structure showed hexameric ring formation. Investigation of the crystal structure of PNT1 revealed presumed active site which was found from other 3?-5? exonuclease homologues. Through mutagenesis of four residues in the presumed active site, Glu52 was identified as a key residue for its 3?-5? exonuclease activity.

Author dc:creator

• Sung, Min Woo 1980-

Contributors dc:contributor

• Li, Pingwei

• Polynucleotidyl transferase
• AIM2

Handle dc:identifier.uri

http://hdl.handle.net/1969.1/148379

OAI identifier oai:identifier

oai:tdl-ir.tdl.org:1969.1/148379