Coupled kinetics of ATP and peptide hydrolysis by Escherichia coli FtsH protease.

FtsH from Escherichia coli is an ATP- and Zn(2+)-dependent integral membrane protease that is involved in the degradation of regulatory proteins such as sigma(32) and uncomplexed subunits of membrane protein complexes such as secY of the protein translocase. We describe a protocol for solubilizing the recombinant enzyme from inclusion bodies and its subsequent refolding and purification to near homogeneity. This is a high-yield protocol and produces in excess of 20 mg of purified FtsH per liter of E. coli culture. We found that refolded FtsH has biochemical properties similar to detergent extracted overexpressed protein described previously. FtsH forms a large complex with an apparent mass of 1200 kDa as determined by gel filtration. Both ATPase and protease activities are coincident with this large complex; smaller forms of FtsH do not exhibit either activity. While FtsH-catalyzed hydrolysis of ATP can occur in the absence of protein substrate (k(c) = 22 min(-1); K(m) = 23 microM), proteolysis shows an absolute dependence on nucleoside-5'-triphosphates, including ATP, CTP, and various analogues. In the presence of 5 mM ATP, FtsH catalyzes the hydrolysis of sigma(32) with the following observed kinetic parameters: k(c) = 0.18 min(-1) and K(m) = 8.5 microM. Significantly, this reaction is processive and generates no intermediate species, but rather, approximately 10 peptide products, all of MW <3 kDa. FtsH protease also efficiently hydrolyzes the peptide Phe-Gly-His-(NO)2Phe-Phe-Ala-Phe-OMe. Hydrolysis occurs exclusively at the (NO)2Phe-Phe bond (k(c) = 2.1 min(-1); K(m) = 12 microM), and like proteolysis, shows an absolute dependence on NTPs. We propose a mechanism for the coupled hydrolytic activities of FtsH toward ATP and peptide substrates that is consistent with a recently proposed structural model for FtsH.

Generation of multiple farnesoid-X-receptor isoforms through the use of alternative promoters.

Bile acid biosynthesis is regulated by both feed-forward and feedback mechanisms involving a cascade of nuclear hormone receptors. Feed-forward regulation of the rate limiting enzyme in bile acid biosynthesis is provided by oxysterols through liver-X-receptor alpha (NR1H3), while feedback regulation is provided by bile acids through farnesoid-X-receptor (FXR) (NR1H4). The Syrian golden hamster provides a useful model for studying lipid metabolism. The hamster metabolizes and transports dietary cholesterol in a similar manner to humans, with the resulting lipid profile being more similar to the human profile than that of other rodent models. Cloning of Fxr from Syrian golden hamster revealed four hamster Fxr splice variants that altered the N-terminal activation domain or the hinge region between the DNA and ligand binding domains. Human genomic sequence and data from hamster Fxr were used to identify and clone a novel human FXR isoform resulting from the use of an alternative promoter. RNA expression analysis indicates that the two human FXR isoforms are differentially expressed in developmental and tissue-specific patterns and are likely to provide a mechanism for cell-specific FXR-dependent transcriptional activity.