ABSTRACT
It is now recognized that post-transcriptional mechanisms are pivotal to renin production. These involve factors that modulate renin mRNA stability. In 2003 new data has emerged from work in Australia and Germany that has identified several of the, as many as, 20 or so proteins involved. These include CP1 (hnRNP E1), HuR, HADHB, dynamin, nucleolin, YP-1, hnRNP K and MINT-homologous protein. Cyclic AMP (cAMP) is a crucial regulator of renin secretion as well as transcriptional and post-transcriptional control of expression. Many of the RNA-binding proteins that were identified responded to forskolin, increasing in amount by two to 10-fold. The cAMP mechanisms that regulate renin mRNA target, at least in large part, other genes that presumably encode some of these proteins. The increase in the expression of these then facilitates, sequentially, renin mRNA stabilization and destabilization. Our data, using a battery of different techniques, confirm that CP1 and HuR stabilize renin mRNA, whereas HADHB causes destabilization. These proteins target cis-acting C-rich sequences (in the case of CP1) and AU-rich sequences (HuR) in the distal region of the 3'-untranslated region of renin mRNA. We found HADHB was enriched in juxtaglomerular cells and that that within Calu-6 cells HADHB, HuR and CP1 all localized in nuclear subregions, as well as cytoplasm (HADHB and CP1) and mitochondria (HADHB) commensurate with the role each plays in control of renin mRNA stability. The specific proteins that bind to human renin mRNA have begun to be revealed. Cyclic AMP upregulates the binding of several of these proteins, which in turn affect renin mRNA stability and thus overall expression of renin.
Subject(s)
Cyclic AMP/physiology , RNA Stability/physiology , RNA-Binding Proteins/physiology , Renin/genetics , Humans , RNA, Messenger/genetics , Renin/biosynthesisABSTRACT
The role of the 13 histidine residues in plasminogen activator inhibitor 1 (PAI-1) for the stability of the molecule was studied by replacing these residues by threonine, using site-directed mutagenesis. The generated mutants were expressed in Escherichia coli, purified and characterized. All variants had a normal activity and formed stable complexes with tissue-type plasminogen activator. Most of these PAI-1 variants displayed a similar pH-dependency in stability as wild-type PAI-1, with increased half-lives at lower pH. However, the variant His364Thr had a half-life of about 50 min at 37 degrees C and had almost completely lost its pH-dependency. Therefore, our data suggest that His(364), in the COOH-terminal end of the molecule might be responsible for the pH-dependent stability of PAI-1.
Subject(s)
Plasminogen Activator Inhibitor 1/chemistry , Amino Acid Substitution , Escherichia coli , Histidine , Humans , Plasminogen Activator Inhibitor 1/genetics , Point Mutation , Serine Proteinase Inhibitors/chemistry , Serine Proteinase Inhibitors/genetics , Structure-Activity RelationshipABSTRACT
Using site-directed mutagenesis, His(143) on the alpha-helix F of PAI-1 was substituted by Lys, Asp, Phe and Thr, respectively. The generated single-site changed plasminogen activator inhibitor-1 (PAI-1) mutants were expressed in Escherichia coli and purified by heparin-Sepharose and anhydrotrypsin agarose chromatographies. When compared with wild-type (wtPAI-1), the PAI-1 mutants His143Asp and His143Phe had shorter half-lives at pH 7.5 (1.1 and 1.4 h, respectively, vs. 2 h for wtPAI-1). They also exhibited less pH dependency of their stability, with half-lives at pH 5.5 of 2.5 and 2.2 h, respectively, vs. 18 h for wtPAI-1. However, the PAI-1 mutants His143Lys and His143Thr had similar properties as wtPAI-1 in this respect. In conclusion, our results suggest that His(143) in one way or another might be involved in the pH-dependent stability of PAI-1. However, it seems that the protonation of this particular residue is of less importance. The PAI-1 mutants His143Asp and His143Phe only displayed about 20% of the specific activity obtained for wtPAI-1, because they, to a large extent, act as substrates for tissue-type plasminogen activator.
Subject(s)
Histidine/chemistry , Plasminogen Activator Inhibitor 1/chemistry , Serine Proteinase Inhibitors/chemistry , Drug Stability , Hydrogen-Ion Concentration , Mutagenesis, Site-Directed , Mutation , Plasminogen Activator Inhibitor 1/genetics , Tissue Plasminogen Activator/antagonists & inhibitorsABSTRACT
Osteogenesis of the body of the mandible in embryonic and neonatal rats was studied histologically and by histochemistry to determine the role of Meckel's cartilage in bone formation. Meckel's cartilage showed intense activity of lactate dehydrogenase and NADH2-diaphorase and weak activity of acid phosphatase, indicating a functioning citric acid cycle, pentose phosphate shunt and a capacity for anaerobic metabolism. The activity of these enzymes declined after hypertrophy of Meckel's cartilage. Alkaline phosphatase was the major enzyme of mineralising mandibular osteoid and was present in the osteoblasts and osteoprogenitor cells but not in Meckel's cartilage. After the differentiation of Meckel's cartilage and intramembranous bone, Meckel's cartilage supported mandibular bone formation by endochondral ossification in the anterior part of the mandible.
Subject(s)
Cartilage/embryology , Mandible/embryology , Alkaline Phosphatase/analysis , Animals , Cartilage/enzymology , Microscopy , Rats , Rats, Inbred StrainsABSTRACT
Type MM phosphoglycerate mutase from free dissected mandibular processes from embryonic rats was reversibly inactivated by tetrathionate, p-chloromercuribenzoate, and Hg2+. Titration with p-chloromercuribenzoate showed the existence of two sulfhydryl groups per enzyme subunit, the modification of which produced a progressive decline in enzyme activity. The apparent Km values for substrate and cofactor were not affected by tetrathionate treatment. Phosphoglycerate mutase inactivated by tetrathionate and by p-chloromercuribenzoate was unable to form the functionally active phosphorylenzyme when mixed with glycerate-2,3-P2. Glycerate-2,3-P2 protected against tetrathionate but failed to protect against Hg2+ and p-chloromercuribenzoate.
Subject(s)
Face/embryology , Isoenzymes/antagonists & inhibitors , Phosphoglycerate Mutase/antagonists & inhibitors , Phosphotransferases/antagonists & inhibitors , Sulfhydryl Compounds/pharmacology , Animals , Chloromercuribenzoates/pharmacology , Embryo, Mammalian , Facial Bones/embryology , Facial Bones/enzymology , Facial Muscles/embryology , Facial Muscles/enzymology , Rats , Rats, Inbred Strains , Spectrophotometry , Tetrathionic Acid/pharmacologyABSTRACT
To characterize the three phosphoglycerate mutase (PGM) isoenzymes present in rat facial processes (types MM, BB, and MB), their sensitivity to reagents of the sulfhydryl groups and to heat treatment has been studied. Type BB PGM was not affected by the -SH group reagents; type MB PGM was inhibited about 50%, and type MM PGM was fully inhibited. Type MB PGM showed a greater heat lability than type MM PGM. There was a developmental change from type BB PGM from the 9th embryonic day to isoenzymes MB and MM on the 15th embryonic day. Isoenzyme development was first seen in mandibular processes, followed by maxillary, lateral nasal, and medial nasal processes.