资源环境科技发展态势分析平台

Cryo-electron microscopy structures of the ATP-permeable channel pannexin 1 reveal a gating mechanism involving multiple distinct ion-conducting pathways.

Pannexin 1 (PANX1) is an ATP-permeable channel with critical roles in a variety of physiological functions such as blood pressure regulation(1), apoptotic cell clearance(2) and human oocyte development(3). Here we present several structures of human PANX1 in a heptameric assembly at resolutions of up to 2.8 angstrom, including an apo state, a caspase-7-cleaved state and a carbenoxolone-bound state. We reveal a gating mechanism that involves two ion-conducting pathways. Under normal cellular conditions, the intracellular entry of the wide main pore is physically plugged by the C-terminal tail. Small anions are conducted through narrow tunnels in the intracellular domain. These tunnels connect to the main pore and are gated by a long linker between the N-terminal helix and the first transmembrane helix. During apoptosis, the C-terminal tail is cleaved by caspase, allowing the release of ATP through the main pore. We identified a carbenoxolone-binding site embraced by W74 in the extracellular entrance and a role for carbenoxolone as a channel blocker. We identified a gap-junction-like structure using a glycosylation-deficient mutant, N255A. Our studies provide a solid foundation for understanding the molecular mechanisms underlying the channel gating and inhibition of PANX1 and related large-pore channels.

Shugoshin and MAD2 regulate separase-mediated chromosome separation during mitosis, in parallel to a previously identified mechanism involving the anaphase inhibitor securin.

Separation of eukaryotic sister chromatids during the cell cycle is timed by the spindle assembly checkpoint (SAC) and ultimately triggered when separase cleaves cohesion-mediating cohesin(1-3). Silencing of the SAC during metaphase activates the ubiquitin ligase APC/C (anaphase-promoting complex, also known as the cyclosome) and results in the proteasomal destruction of the separase inhibitor securin(1). In the absence of securin, mammalian chromosomes still segregate on schedule, but it is unclear how separase is regulated under these conditions(4,5). Here we show that human shugoshin 2 (SGO2), an essential protector of meiotic cohesin with unknown functions in the soma(6,7), is turned into a separase inhibitor upon association with SAC-activated MAD2. SGO2-MAD2 can functionally replace securin and sequesters most separase in securin-knockout cells. Acute loss of securin and SGO2, but not of either protein individually, resulted in separase deregulation associated with premature cohesin cleavage and cytotoxicity. Similar to securin(8,9), SGO2 is a competitive inhibitor that uses a pseudo-substrate sequence to block the active site of separase. APC/C-dependent ubiquitylation and action of the AAA-ATPase TRIP13 in conjunction with the MAD2-specific adaptor p31(comet) liberate separase from SGO2-MAD2 in vitro. The latter mechanism facilitates a considerable degree of sister chromatid separation in securin-knockout cells that lack APC/C activity. Thus, our results identify an unexpected function of SGO2 in mitotically dividing cells and a mechanism of separase regulation that is independent of securin but still supervised by the SAC.