1. TgOTUD3A selectively deubiquitinates K48-linked Toxoplasma proteins.
TgOTUD3A selectively deubiquitinates K48-linked Toxoplasma proteins. Washed parasites were mechanically lysed and incubated in the DUB reaction buffer without (control) or with either recombinant wild-type TgOTUD3A or mutant TgOTUD3A (C229A) enzyme in a 30-min reaction, resolved using SDS-PAGE, transferred, and probed with linkage-specific antibodies. (A) K48 linkage-specific antibody shows the removal of K48-linked ubiquitin T. gondii proteins in the presence of the wild type but not its cysteine (C229A) mutant. Notably, all K48-linked ubiquitin modifications are removed from protein targets by the wild-type enzyme. (B) The same enzyme mix used in panel A was tested using the synthetic K48 polyubiquitin chains, confirming activity in the wild-type enzyme and no activity in the C229A mutant. (C) The same parasite extract and enzyme used in panel A were resolved and probed with a K63 linkage-specific antibody. Neither wild-type nor mutant enzyme was able to digest T. gondii K63-linked ubiquitinated proteins, confirming that K63-linked polyubiquitin is a poor substrate for TgOTUD3A. (D) Enzyme and buffer controls using the synthetic K63-linked substrate confirm partial activity. Both digestion blots were stripped and reprobed with TgSAG1 antibody for a loading control (A and C).
Animesh Dhara, and Anthony P. Sinai mSphere 2016;1:e
Copyright © 2016 Dhara and Sinai.