Regulation of human separase by securin binding and autocleavage.
BACKGROUND
Sister chromatid separation is initiated by separase, a protease that cleaves cohesin and thereby dissolves sister chromatid cohesion. Separase is activated by the degradation of its inhibitor securin and by the removal of inhibitory phosphates. In human cells, separase activation also coincides with the cleavage of separase, but it is not known if this reaction activates separase, which protease cleaves separase, and how separase cleavage is regulated.


RESULTS
Inhibition of separase expression in human cells by RNA interference causes the formation of polyploid cells with large lobed nuclei. In mitosis, many of these cells contain abnormal chromosome plates with unseparated sister chromatids. Inhibitor binding experiments in vitro reveal that securin prevents the access of substrate analogs to the active site of separase. Upon securin degradation, the active site of full-length separase becomes accessible, allowing rapid autocatalytic cleavage of separase at one of three sites. The resulting N- and C-terminal fragments remain associated and can be reinhibited by securin. A noncleavable separase mutant retains its ability to cleave cohesin in vitro.


CONCLUSIONS
Our results suggest that separase is required for sister chromatid separation during mitosis in human cells. Our data further indicate that securin inhibits separase by blocking the access of substrates to the active site of separase. Securin proteolysis allows autocatalytic processing of separase into a cleaved form, but separase cleavage is not essential for separase activation.
Research Institute of Molecular Pathology, Dr. Bohr-Gasse 7, 1030, Vienna, Austria.
Irene
I
Waizenegger
Juan F
JF
Giménez-Abián
Dominik
D
Wernic
Jan-Michael
JM
Peters
D000595:Amino Acid Sequence; D000818:Animals; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone; D002875:Chromosomes / Q000378:metabolism*; D010450:Endopeptidases; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008938:Mitosis / Q000235:genetics; D008954:Models, Biological; D015394:Molecular Structure; D009363:Neoplasm Proteins / Q000378:metabolism*; D009687:Nuclear Proteins; D010455:Peptides / Q000378:metabolism; D010750:Phosphoproteins; D011123:Polyploidy; D011485:Protein Binding; D034622:RNA Interference; D029701:Saccharomyces cerevisiae Proteins; D064249:Securin; D064247:Separase; D016415:Sequence Alignment; D015534:Trans-Activators / Q000378:metabolism*
D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; D004791:Enzyme Inhibitors; D005656:Fungal Proteins; C490529:MCD1 protein, S cerevisiae; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D010455:Peptides; D010750:Phosphoproteins; D029701:Saccharomyces cerevisiae Proteins; D064249:Securin; D015534:Trans-Activators; C578002:pituitary tumor-transforming protein 1, human; D010450:Endopeptidases; C577986:ESP1 protein, S cerevisiae; C577987:ESPL1 protein, human; D064247:Separase