Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.9 (enterokinase)
 675 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Bone morphogenetic proteins (BMPs) belong to the transforming growth factor-beta (TGF-beta) superfamily of multifunctional cytokines. BMP induces its signal to regulate growth, differentiation, and apoptosis of various cells upon trimeric complex formation with two distinct type I and type II receptors on the cell surface: both are single-transmembrane serine/threonine kinase receptors. To identify the amino acid residues on BMP type I receptor responsible for its ligand binding, the structure-activity relationship of the extracellular ligand-binding domain of the BMP type IA receptor (sBMPR-IA) was investigated by alanine-scanning mutagenesis. The mutant receptors, as well as sBMPR-IA, were expressed as fusion proteins with thioredoxin in Escherichia coli, and purified using reverse phase high performance liquid chromatography (RP-HPLC) after digestion with enterokinase. Structural analysis of the parent protein and representative mutants in solution by CD showed no detectable differences in their folding structures. The binding affinity of the mutants to BMP-4 was determined by surface plasmon resonance biosensor. All the mutant receptors examined, with the exception of Y70A, displayed reduced affinities to BMP-4 with the rank order of decreases: I52A (17-fold) approximately F75A (15-fold) >> T64A (4-fold) = T62A (4-fold) approximately E54A (3-fold). The decreases in binding affinity observed for the latter three mutants are mainly due to decreased association rate constants while alterations in rate constants both, for association and dissociation, result in the drastically reduced affinities for the former two mutants. These results allow us to conclude that sBMPR-IA recognizes the ligand using the concave face of the molecule. The major ligand-binding site of the BMP type IA receptor consists of Phe75 in loop 2 and Ile52, Glu54, Thr62 and Thr64 on the three-stranded beta-sheet. These findings should provide a general basis for the ligand/type I receptor recognition in the TGF-beta superfamily.
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PMID:Identification of the ligand-binding site of the BMP type IA receptor for BMP-4. 1124 Dec 15

The transforming growth factor-beta (TGF-beta) superfamily member, activin A, plays a central role in the regulation of multiple physiological processes including cell differentiation, mitogenesis, embryogenesis, apoptosis and inflammation. In normal cells, activin A signalling is regulated to maintain cellular and tissue health and suppress tumour growth. Disruption of activin A signalling has been implicated in tumour formation and progression. Hence, the availability of activin A is an important target for the development of diagnostics and drugs for therapeutic intervention. To this end, we have expressed human activin A in Pichia pastoris, permitting its secretion into culture medium and purification as the mature homodimer. A construct was engineered encoding the monomeric precursor protein with a N-terminal FLAG affinity tag (DYKDDDDK) and a cleavage site (EKR) for Kex2p protease. Procedures for the two-step purification of human activin A by ion-exchange and anti-FLAG antibody affinity chromatography, and for the removal of the FLAG affinity tag from purified recombinant human activin A by enteropeptidase, are described. The molecular weights of the FLAG-tagged and de-tagged human activin A were confirmed by MALDI-TOF mass spectroscopy. The biological activity of these recombinant activins was assessed for their effects on modulating the secretion of Endothelin-1 (ET-1) by human umbilical vein endothelial cells (HUVECs). The recombinant human activin A containing the intact FLAG tag resulted in a reduced ET-1 secretion from HUVECs, whereas upon removal of this affinity purification tag the purified recombinant human activin A restored ET-1 secretion to levels comparable to the positive control. These results document an approach of considerable potential for the simple, large-scale expression and purification of this important human growth factor for use in diagnostic and therapeutic purposes.
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PMID:Synthesis, purification and bioactivity of recombinant human activin A expressed in the yeast Pichia pastoris. 1902 59