Evading the proteasome: absence of lysine residues contributes to pertussis toxin activity by evasion of proteasome degradation.
Pertussis toxin (PT) is an important virulence factor produced by Bordetella pertussis. PT holotoxin comprises one enzymatically active A subunit (S1), associated with a pentamer of B subunits. PT is an ADP-ribosyltransferase that modifies several mammalian heterotrimeric G proteins. Some bacterial toxins are believed to undergo retrograde intracellular transport through the Golgi apparatus to the endoplasmic reticulum (ER). The ER-associated degradation (ERAD) pathway involves the removal of misfolded proteins from the ER and degradation upon their return to the cytosol; this pathway may be exploited by PT and other toxins. In the cytosol, ERAD substrates are ubiquitinated at lysine residues, targeting them to the proteasome for degradation. We hypothesize that S1 avoids ubiquitination and proteasome degradation due to its lack of lysine residues. We predicted that the addition of lysine residues would reduce PT toxicity by allowing ubiquitination and degradation to occur. Variant forms of PT were engineered, replacing one, two, or three arginines with lysines in a variety of locations on S1. Several variants were identified with wild-type in vitro enzymatic activity but reduced cellular activity, consistent with our hypothesis. Significant recovery of the cellular activity of these variants was observed when CHO cells were pretreated with a proteasome inhibitor. We concluded that the replacement of arginine residues with lysine in the S1 subunit of PT renders the toxin subject to proteasomal degradation, suggesting that wild-type PT avoids proteasome degradation due to an absence of lysine residues.
Worthington
Zoë E V
ZE
Department of Microbiology and Immunology, University of Maryland School of Medicine, 660 W. Redwood Street, Baltimore, MD 21201, USA. zphil001@umaryland.edu
Carbonetti
Nicholas H
NH
D000818:Animals; D001886:Bordetella pertussis / Q000235:genetics / Q000472:pathogenicity*; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D017353:Gene Deletion; D008239:Lysine / Q000378:metabolism; D037342:Pertussis Toxin / Q000737:chemistry / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D011489:Protein Denaturation
C083388:pertussis toxin, S1 subunit; D037342:Pertussis Toxin; D046988:Proteasome Endopeptidase Complex; D008239:Lysine