Inhibitory domains

The human PR exists as two functionally distinct isoforms PRA and PRB transcribed from two promoters from a single gene. PRA lacks the N-terminal 164 aa and is a 769 aa protein. PRB functions as a transcriptional activator in most cell and promoter contexts. In contrast, PRA is transcriptionally inactive and functions as a strong ligand-dependent transdominant repressor of SHR transcriptional activity. Different cofactor interactions were demonstrated for PRA and PRB, probably due to an inhibitory domain within the first 140 aa of PRA, which is masked in PRB. Both PR isoforms however, repress estradiol-induced ER activity when liganded. Several other mRNA isoforms are present in PR-positive tissues such as breast cancer with unknown clinical significance. 

Predicted secondary structure of tissue factor pathway inhibitor (TFPI) showing the Factor Xa and Factor Vila inhibitory domains. The arrows point to the PI sites. 

Fig. 12-23). Glycogen synthase kinase 3 (GSK3) is inactive when phosphorylated on a Ser residue in its auto-inhibitory domain (Fig. 12-23b). Dephosphorylation of that domain frees the enzyme to bind and phosphory-late its target proteins. Similarly, the polar head group of the phospholipid PIP3, protruding from the inner leaflet of the plasma membrane, provides points of attachment for proteins that contain SH3 and other domains. 

Antigen unmasking on sections of paraffin-embedded tissues can be accomplished by reduction of disulfide bonds by treatment with 2-mercaptoethanol, followed by alkylation with sodium iodoacetate to prevent the bonds from reforming. This method has been used for unmasking a Kunitz protease inhibitory domain epitope of Alzheimer s amyloid precursor protein in human brain (Campbell et al., 1999). Sections are reduced with a mixture of 0.14 M 2-mercaptoethanol in 0.5 M Tris-HCl (pH 8.0) and 1 mM EDTA for 3 hr in the dark at room temperature. After being washed for 3 min in distilled water, the sections are treated with a mixture of 250 mg/ml iodoacetic acid in 0.1 M NaOH, diluted 1 10 in 0.5 M Tris-HCl (pH 8.0) and 1 mM EDTA for 20 min in the dark. 

Girard TJ, Warren LA, Novotny Wp Likert KM, Brown SG, Miletich JR et al. Functional significance of the Kunitz-type inhibitory domains of lipoprotein-associated coagulation inhibitor. Nature 1989 338 518-520. 

The cereal dual function a-amylase/trypsin inhibitor proteins are cysteine-rich, disulphide-rich, double-headed, 13-16 kDa, dual function inhibitor proteins that inhibit both of the digestion enzymes a-amylase and trypsin [290-325] (Table 11). Thus the Zea (com) member of this family, com Hageman factor inhibitor (CHFI), is a double-headed 14 kDa protein that inhibits a-amylase and the serine proteases trypsin and blood clotting Factor Xlla [323-324] (Table 11). The structures of the bifunctional a-amylase/trypsin inhibitor proteins from Eleusine (ragi) (RBI) [292-295] and Zea (com) (CHFI) [325] have been determined. These proteins are structurally similar to the lipid transfer proteins, being composed of a bundle of 4 a-helices together with a short [3-sheet element connected by loops, the a-amylase- and protease-inhibitory domains being separately located [325]. 

Solanum americanum (Solanaceae) [phloem] SaPIN2a SaPIN2b (13 kDa 2 inhibitory domains) (processed SaPIN2a 121 aa 13 kDa 8 Cys 4 S-S) Chymotrypsin (F62-E63, putative) Trypsin (R5-E6, putative) [500]... 

Morishita H, Yamakawa T, Matsusue T, Kusuyama T, Sameshima-Aruga R, Hirose J, et al. Novel factor Xa and plasma kallikrein inhibitory activities of the second Kunitz-type inhibitory domain of urinary trypsin inhibitor. Thromb Res 1994 73 193-204. 

A family of homologous protein kinase C isoenzymes (e.g. PKC-a, (3, y, 8, and T ) are variously activated by Ca2+, phospholipids (notably phosphatidylserine) and diacylglycerol (DAG). The inactive PKC is autoinhibited by an inhibitory domain and binding of the activating ligands changes the conformation of the autoinhibitory domain in a subtle way that overcomes the inhibition. 

Trypsin cleaves a peptide bond on the C-terminal side of a basic residue such as arginine (Arg) or lysine (Lys) whereas chymotrypsin cleaves on the C-terminal side of the hydrophobic residues phenylalanine (Phe), tryptophan (Trp) or tyrosine (Tyr). Elastase cleaves on the C-terminal side of small amino acids such as alanine (Ala) and glycine (Gly). A large number of serine PI proteins have been isolated from plants (Table 13.4) and the substrate specificity of the target proteases corresponds with the inhibitory amino acid sequences (P2-P1-PT-P2 ) of the PI proteins. Thus, the double-headed trypsin- and chymotrypsin-inhibitory Bowman-Birk PI protein 1 (BBI-1) from soybean (Glycine BBI-1, Table 13.5G) has a Pl-PT sequence of Lys—Ser at the trypsin inhibitory domain I site and a PI PI sequence of Leu-Ser at the chymotrypsin inhibitory domain II site. 

Another simple approach for the detection of noncovalent interactions is to compare the peptide maps produced by proteolysis of the target protein and its complex with other ligands. This approach, also known as epitope mapping, relies on the fact that the two maps differ qualitatively because the contact regions of the interacting proteins are shielded from the protease s activity in the complex. An illustrative example is the detection of protein-protein interaction between a protein representing the kinase inhibitory domain of the cell cycle regulatory protein (p21-B) and cyclin-dependent kinase 2 (cdk).164... 

In addition to the metal interactions with APP and Ab that may directly affect the generation of Ab and its aggregation and toxicity, biometals have been found to interact with many of the proteins and activities that surround APP. These interactions are hkely to subserve physiological purposes, and may reflect a role for APP metabolism in metal homeostasis. Zn has been shown to interact with and inhibit the gamma-secretase complex [ 172]. The intracellular carboxyl terminus of APP interacts with XIla (MINT), which in turn interacts with the Cu Chaperone of SODl (CCSl) directing Cu away from SODl [173]. CCSl interacts with a Cu(I)-binding site on the intracellular carboxyl terminus of BACEl [174], although it is not yet clear whether this influences BACE activity. This may be part of the mechanism by which Cu added to cell culture increases Ab release into the culture medium (unpublished data). A metalloproteinase inhibitory domain has been identified in the portion of APP immediately upstream from the Ab domain [175]. 

Stimulatory Activation of c-Src by peroxynitrite-induced nitration of inhibitory domain [58]... 

Lycopersicon esculentum (tomato) (Solanaceae) ARPi (= TR8) (proprotein 223 aa 25 kDa 3x8 Cys 3x4S-S 3 X 64 aa repeated inhibitory domains) Trypsin (K29-E30, repeat 1 K93-E94, repeat 2 K157-E158, repeat 3) [488, 489]... 

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