Gene/Protein
Disease
Symptom
Drug
Enzyme
Compound
Pivot Concepts:
Gene/Protein
Disease
Symptom
Drug
Enzyme
Compound
Target Concepts:
Gene/Protein
Disease
Symptom
Drug
Enzyme
Compound
Query: EC:3.4.24.27 (
thermolysin
)
1,894
document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)
Protease-activated receptors (PARs) are G-protein-coupled receptors that are activated by proteolysis of the N-terminus, which exposes a tethered ligand that interacts with the receptor. Numerous studies have focused on the signaling pathways mediated by PARs. However, the structural basis for initiation of these pathways is unknown. Here, we describe a strategy for the expression and purification of
PAR4
. This is the first PAR family member to be isolated without stabilizing modifications for biophysical studies. We monitored
PAR4
activation with histidine hydrogen-deuterium exchange.
PAR4
has nine histidines that are spaced throughout the protein, allowing a global view of solvent accessible and nonaccessible regions. Peptides containing each of the nine His residues were used to determine the
t
1/2
for each His residue in apo or thrombin-activated
PAR4
. The thrombin-cleaved
PAR4
exhibited a 2-fold increase (
p
> 0.01) in
t
1/2
values observed for four histidine residues (His
180
, His
229
, His
240
, and His
380
), demonstrating that these regions have decreased solvent accessibility upon thrombin treatment. In agreement, thrombin-cleaved
PAR4
also was resistant to
thermolysin
digestion. In contrast, the rate of
thermolysin
proteolysis following stimulation with the
PAR4
activation peptide was the same as that of unstimulated
PAR4
. Further analysis showed the C-terminus is protected in thrombin-activated
PAR4
compared to uncleaved or agonist peptide-treated
PAR4
. The studies described here are the first to examine the tethered ligand activation mechanism for a PAR family member biophysically and shed light on the overall conformational changes that follow activation of PARs by a protease.
...
PMID:Expression and Purification of Protease-Activated Receptor 4 (PAR4) and Analysis with Histidine Hydrogen-Deuterium Exchange. 3195 46
