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Mutational and kinetic analysis of the endosome‐associated deubiquitinating enzyme, AMSH
Author(s) -
Davies Christopher Williamson,
Kim Myung-Il,
Paul Lake N,
Das Chittaranjan
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.754.2
Subject(s) - escrt , deubiquitinating enzyme , ubiquitin , endosome , biology , microbiology and biotechnology , tsg101 , chemistry , biochemistry , gene , intracellular , microrna , microvesicles
The endosomal‐sorting complexes required for transport (ESCRT) is a ubiquitin‐mediated process that is implicated in endosomal sorting, trafficking, and viral budding. The ESCRT machinery regulates signaling events by internalizing cell surface receptors and targeted them for degradation by the lysosome. Two deubiquitinating enzymes (DUBs) regulate the activity of the ESCRT complex, STAM‐binding protein (AMSH), and ubiquitin specific protease Y (UBPY), by counteracting the ubiquitination of the cargo. Recently, we published two crystal structures of the catalytic domain of AMSH, and biochemical and modeling studies revealed that AMSH is significantly different from the ESCRT‐independent, AMSH‐like protein (AMSH‐LP) in its stability and ubiquitin recognition.. In the present study, we sought to understand AMSH's mode of substrate recognition, through mutational analysis. Surprisingly, we have discovered a point mutant that shows higher catalytic activity than the wild‐type enzyme. These results support the idea that AMSH is conformationally plastic, which may be biologically relevant. The mechanism of activation and its implication in the functional role played by AMSH within ESCRT complex will be discussed. Results from our studies could provide further insight into understanding the role of AMSH within the ESCRT complex. Support: NIH (1R01RR026273) and the Indiana Space Grant Consortium